WO2019067592A1 - Traitement et détection d'une infection et d'une maladie associées à différents pathogènes fongiques - Google Patents

Traitement et détection d'une infection et d'une maladie associées à différents pathogènes fongiques Download PDF

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Publication number
WO2019067592A1
WO2019067592A1 PCT/US2018/052923 US2018052923W WO2019067592A1 WO 2019067592 A1 WO2019067592 A1 WO 2019067592A1 US 2018052923 W US2018052923 W US 2018052923W WO 2019067592 A1 WO2019067592 A1 WO 2019067592A1
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peptide
kex
subject
pneumocystis
antibody
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Karen A. Norris
Whitney RABACAL
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University of Georgia Research Foundation Inc
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University of Georgia Research Foundation Inc
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Priority to US16/650,493 priority Critical patent/US11547747B2/en
Priority to EP18861133.9A priority patent/EP3687629A4/fr
Publication of WO2019067592A1 publication Critical patent/WO2019067592A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/10—Antimycotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0002—Fungal antigens, e.g. Trichophyton, Aspergillus, Candida
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/14—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from fungi, algea or lichens
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/60—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • C12N9/62—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi from Aspergillus

Definitions

  • Pathogenic fungal organisms are universal in the environment and can cause global health threats. Pathogenic fungi are typically not harmful to individuals with healthy and normally functional immune systems even after exposure, for example, by inhalation.
  • the invention generally provides methods for treating or preventing infection (e.g., opportunistic infection) by fungal pathogens of different etiologies, as well as treating or preventing pulmonary disease and poor pulmonary function associated with infection by different fungal pathogens.
  • infection e.g., opportunistic infection
  • disease-causing fungal pathogens include Pneumocystis, which colonizes lung tissue and causes severe pneumonia after infection; Aspergillus, a common mold, which causes aspergillosis, allergic reactions, lung infections and other health problems; Candida, which typically reside in the intestinal tract and mucous membranes and can cause thrush, infections and invasive candidiasis upon systemic infection, especially in those in poor health or with weak immune systems; and Cryptococcus, which can infect the lungs where it can cause pneumonia-like illness and the brain, where it can cause meningitis.
  • Pneumocystis which colonizes lung tissue and causes severe pneumonia after infection
  • Aspergillus a common mold, which causes aspergillosis, allergic reactions, lung infections and other health problems
  • Candida which typically reside in the intestinal tract and mucous membranes and can cause thrush, infections and invasive candidiasis upon systemic infection, especially in those in poor health or with weak immune systems
  • Cryptococcus which can infect the
  • the present invention stems from the findings described herein that mammalian subjects, immunized with a peptide derived from the Kexin (Kex or Kexl) protein, a subtilisin-like serine protease, from one type of fungal pathogen, Pneumocystis
  • anti-Kexl antibodies monoclonal or polyclonal antibodies
  • the mammalian subjects were non-human primates such as rhesus macaques.
  • antiserum obtained or isolated from the immunized subjects contained antibodies that immunologically recognized and reacted with Kex peptides of at least two fungal pathogens different from the original Kex peptide immunogen.
  • This antibody-containing antiserum can serve as a treatment for infection and/or disease caused by more than one fungal pathogen and can provide immunity against multiple fungal pathogens in another or unrelated subject (i.e., a recipient subject) who receives the antiserum via a suitable mode and route of administration.
  • a subject from whom an antiserum is obtained or isolated is a "donor subject," and a subject to whom the isolated antiserum is administered or provided is a "recipient subject.”
  • a subject is a mammal, particularly a human being or a non-human primate.
  • a recipient subject may be a patient or an individual in need of treatment for or protection from infection or disease caused by one or more of the Pneumocystis, Candida, Aspergillus and/or
  • the invention provides a method of treating or protecting a subject against infection by a fungal pathogen and/or treating or protecting a subject against a disease or dysfunction (e.g., pulmonary disease, chronic obstructive pulmonary disease (COPD), and/or poor pulmonary function), in which the method comprises administering to a subject (a recipient subject) a peptide immunogen derived from the Kexin protein of a fungal pathogen or an antiserum comprising antibodies produced against the peptide immunogen that is obtained or isolated from a donor subject who has been previously immunized (e.g., inoculation by or exposure to) with a fungal immunogen, e.g., a peptide immunogen derived from the Kexin protein of a fungal pathogen such as Pneumocystis, more specifically, Pneumocystis hominis or Pneumocystis jirovecii (formerly called Pneumocystis carinii).
  • the immunogen was provided or present in an amount effective for the donor subject to generate a humoral immune response in which antibodies produced against the original Kex peptide immunogen also cross-reacted with (had binding specificity for and/or activity against) a Kex peptide immunogen of other fungal pathogens (e.g., fungal organisms of different etiologies.
  • a Kex peptide immunogen of other fungal pathogens e.g., fungal organisms of different etiologies.
  • an antiserum is generated in the donor subject against a Kex protein or peptide thereof as shown, for example, in FIG. 4, such as a Kex peptide of about 88-90 amino acids, derived from the fungal pathogen and used as immunogen.
  • the Kex peptide is the Kexl peptide from a Pneumocystis fungal organism.
  • the immunogen is the Kexl peptide of Pneumocystis hominis or Pneumocystis jirovecii.
  • the Pneumocystis Kexl peptide is a 90 amino acid peptide as isolated from a macaque organism infected with Pneumocystis macctccte (also called a reference sequence herein).
  • the immunogen is a Kexl peptide derived from the Kexin protein of a Candida fungal organism.
  • the immunogen is a Kex2 peptide of Candida albicans. In an embodiment, the Candida Kexl peptide is a 90 amino acid Kexl peptide. In an embodiment, the immunogen is a Candida Kexl peptide of 88 amino acids. In an embodiment, the immunogen is a Kex peptide derived from the KexinB protein of an Aspergillus fungal organism. In a particular embodiment, the immunogen is a Kex peptide of Aspergillus fumigatus. In an embodiment, the Aspergillus Kex peptide is a 90-amino acid KexB peptide.
  • the immunogen is an Aspergillus KexB peptide of 88 amino acids.
  • the immunogen is a Kex peptide derived from the Kexin protein of a Cryptococcus fungal organism.
  • the immunogen is a Kex peptide of Cryptococcus neoformans.
  • the Cryptococcus Kex peptide is a 90-amino acid Kex peptide.
  • the immunogen is a Cryptococcus Kex peptide of 117 amino acids.
  • the Kexl peptide is recombinantly produced.
  • the amino acid sequences of the above noted peptides are provided herein, for example, as set forth in FIG. 3.
  • the invention provides a method of treating or protecting a subject against infection by one or more fungal pathogens and/or treating or protecting a subject against disease or dysfunction caused by infection by one or more fungal pathogens (e.g., pulmonary disease, chronic obstructive pulmonary disease (COPD), pneumonia, or poor pulmonary function), in which the method comprises administering to a subject in need thereof a peptide immunogen derived from the Kexin protein of a fungal pathogen or an isolated antiserum containing one or more antibodies directed against an immunogen, e.g., a Kex peptide immunogen derived from the Kexin protein of a fungal pathogen such as Pneumocystis, more specifically, Pneumocystis hominis or Pneumocystis carinii, in an amount effective for the subject to be immunologically protected against infection and/or disease caused by a different (unrelated) fungal pathogen.
  • the different (unrelated) fungal pathogen comprises a non-identical Kex peptide).
  • antibodies in the isolated antiserum cross-react with (have binding specificity for and activity against) a Kex peptide of one or more, particularly, two or more different fungal pathogens.
  • the method advantageously affords treatment or prevention of infection and/or disease caused by or associated with more than one fungal type, for example, one or more of Pneumocystis, Aspergillus, Candida and Cryptococcus fungal organisms.
  • the isolated antiserum is present in a composition, i.e., an immunogenic composition that affords protection against two or more distinct fungal pathogens in the subject receiving the composition following administration.
  • the invention provides a method of generating a cross- protective immune response against different fungal pathogens, e.g., Pneumocystis,
  • the method involves administering to a subject (i.e., a recipient subject) an antiserum obtained or isolated from a donor subject who has previously been inoculated with, exposed to, or administered an immunogen comprising a Kexin polypeptide or an immunogenic peptide portion thereof from a fungal pathogen in an amount effective for the donor subject to produce antibodies directed against the Kex polypeptide or peptide immunogen, which antibodies also recognize and bind to a Kexin polypeptide or a peptide portion thereof from a different fungal pathogen.
  • a subject i.e., a recipient subject
  • an antiserum obtained or isolated from a donor subject who has previously been inoculated with, exposed to, or administered an immunogen comprising a Kexin polypeptide or an immunogenic peptide portion thereof from a fungal pathogen in an amount effective for the donor subject to produce antibodies directed against the Kex polypeptide or peptide immunogen, which antibodies also recognize and bind to a Kexin polypeptide or
  • the antibodies are constituents of the donor subject's blood serum or plasma, which is understood to be an antiserum (immune serum) or immune plasma that can be isolated and employed as treatment or therapy to treat or protect another (unrelated) subject against infection or disease caused by multiple fungal pathogens which comprise a Kex peptide that is specifically targeted by one or more anti-Kex peptide antibodies in the antiserum.
  • the antiserum is an isolated antiserum.
  • the immune plasma is isolated immune plasma.
  • the isolated antiserum may be processed, e.g., concentrated, diluted in a suitable diluent or excipient, chromatographed, purified, e.g., via affinity chromatography, using procedures practiced by one of skill in the art, prior to its use as a therapeutic in the methods described herein.
  • the antibodies administered to a recipient subject may be naturally occurring anti-Kex protein or peptide antibodies that are isolated and purified from another subject who has been naturally exposed (e.g., nonimmunized) to one or more, two or more, or three or more of the fungal pathogens and has consequently raised an anti-Kex protein or peptide antibody immune response against Kex protein or peptide as immunogen.
  • the exposed subject has generated an immune response (and/or a memory B cell response) comprising monoclonal and/or polyclonal antibodies directed against one or more of the Pneumocystis, Aspergillus, Candida and Cryptococcus fungal pathogens.
  • the anti-Kex protein or peptide antibodies isolated (and purified) from the nonimmunized subject are monoclonal antibodies (e.g., directed against the Kex protein or peptide of one of Pneumocystis, Aspergillus, Candida and Cryptococcus).
  • the anti-Kex protein or peptide antibodies isolated (and purified) from the nonimmunized subject are polyclonal antibodies (e.g., directed against the Kex protein or peptide of one or more of Pneumocystis, Aspergillus, Candida and Cryptococcus).
  • the antibodies generated by the practice of the described methods constitute a cross-reactive antiserum (e.g., an isolated antiserum), or naturally occurring antifungal Kex protein or peptide antibodies isolated and purified from a nonimmunized subject as described above, which may be used as a therapeutic or prophylactic treatment against infection and/or disease or the symptoms thereof caused by one or more fungal pathogens having Kexin polypeptides or a Kex peptide portions thereof that are non-identical (show sequence variability) to the Kexin protein or peptide used as immunogen.
  • a cross-reactive antiserum e.g., an isolated antiserum
  • naturally occurring antifungal Kex protein or peptide antibodies isolated and purified from a nonimmunized subject as described above which may be used as a therapeutic or prophylactic treatment against infection and/or disease or the symptoms thereof caused by one or more fungal pathogens having Kexin polypeptides or a Kex peptide portions thereof that are non-identical
  • the subject is immunized with a Kex peptide derived from Pneumocystis or Pneumocystis jirovecii.
  • the isolated antiserum contains one or more antibodies generated against a Kex peptide derived from Pneumocystis or Pneumocystis jirovecii, in which the one or more antibodies are cross-reactive (have multi-specificity for) and recognize (e.g., bind to and/or neutralize) a Kex peptide of one or more of Candida or Candida albicans, Aspergillus or Aspergillus fumigatus, or Cryptococcus or Cryptococcus neoformans.
  • the isolated antiserum contains one or more antibodies that are generated against a Kex peptide of one or more of Candida or Candida albicans, Aspergillus or Aspergillus fumigatus, or Cryptococcus or Cryptococcus neoformans and are also cross-reactive with (are multi-specific for) a Kex peptide of Pneumocystis or
  • the naturally occurring anti -fungal Kex protein or peptide antibodies isolated and purified from a nonimmunized subject are monoclonal or polyclonal antibodies directed against the Kex protein or peptide of one, or more than one of Pneumocystis or Pneumocystis jirovecii, Candida or Candida albicans, Aspergillus or Aspergillus fumigatus, or Cryptococcus or Cryptococcus neoformans, as described herein.
  • the invention provides a method of treating or preventing a disease or symptoms thereof associated with infection by a Candida fungal pathogen, in which the method comprises administering to a subject in need thereof an immunologically effective amount of an isolated antiserum, or monoclonal or polyclonal antibodies isolated and purified from a nonimmunized subject, and generated against a Kexl peptide of Pneumocystis or Pneumocystis jirovecii.
  • the fungal pathogen is Candida albicans.
  • the antiserum contains antibodies generated against the 90-amino acid
  • Pneumocystis Kexl peptide which antibodies and antiserum are generated in a subject immunized with the Pneumocystis Kexl peptide.
  • the isolated antiserum is administered in a pharmaceutically acceptable composition.
  • the invention provides a method of treating or preventing a disease or symptoms thereof associated with infection by an Aspergillus fungal pathogen, in which the method comprises administering to a subject in need thereof an immunologically effective amount of an isolated antiserum, or monoclonal or polyclonal antibodies isolated and purified from a nonimmunized subject, generated against a Kexl peptide of Pneumocystis or Pneumocystis jirovecii.
  • the fungal pathogen is Aspergillus fumigatus.
  • the antiserum contains antibodies generated against the 90-amino acid Pneumocystis Kexl peptide, which antibodies and antiserum are generated in a subject immunized with the Pneumocystis Kexl peptide.
  • the isolated antiserum is administered in a pharmaceutically acceptable composition.
  • the invention provides a method of treating or preventing a disease or symptoms thereof associated with infection by a Cryptococcus fungal pathogen, in which the method comprises administering to a subject in need thereof an immunologically effective amount of an isolated antiserum, or monoclonal or polyclonal antibodies isolated and purified from a nonimmunized subject, generated against a Kexl peptide of Pneumocystis or
  • the fungal pathogen is Cryptococcus neoformans.
  • the antiserum contains antibodies generated against the 90-amino acid Pneumocystis Kexl peptide (FIG. 3), which antibodies and antiserum are generated in a subject immunized with the Pneumocystis Kexl peptide.
  • the isolated antiserum is administered in a pharmaceutically acceptable composition.
  • the antiserum e.g., isolated antiserum
  • monoclonal or polyclonal antibodies isolated and purified from a
  • nonimmunized subject comprising anti-Kex peptide antibodies can treat or protect a subject against infection and disease, such as pulmonary disease, pneumonia, or fungal colonization in lung or brain tissue, caused by one or more different fungal pathogens.
  • the antiserum isolated antiserum
  • the antiserum comprising mti-Pneumocystis hominis or Pneumocystis jirovecii Kexl peptide antibodies is used in the treatment of disease or infection of a subject who is infected by, or who is at risk of infection by, a fungal organism selected from Aspergillus fumigates, Candida albicans and Cryptococcus neoformans.
  • the invention provides a method of immunizing a subject to treat or prevent disease resulting from infection by one or more fungal pathogens, comprising administering to the subject an antiserum (e.g., an isolated antiserum), or monoclonal or polyclonal antibodies isolated and purified from a nonimmunized subject, comprising antibodies that specifically target and bind to two or more of a Pneumocystis Kex protein or peptide, an Aspergillus, Candida or Cryptococcus Kex protein or peptide.
  • the method elicits a B-cell memory response for protection against subsequent exposure to one or more of the fungal pathogens.
  • the methods are useful for treating and protecting against pulmonary (lung) disease, such as chronic obstructive pulmonary disease (COPD), including, without limitation, bronchitis, chronic bronchitis, emphysema, asthma including severe asthma (e.g., refractory (non-reversible asthma)), or bronchiectasis.
  • lung disease such as chronic obstructive pulmonary disease (COPD)
  • COPD chronic obstructive pulmonary disease
  • bronchitis bronchitis
  • chronic bronchitis chronic bronchitis
  • emphysema emphysema
  • severe asthma e.g., refractory (non-reversible asthma)
  • bronchiectasis bronchiectasis
  • the methods are useful for treating or protecting against colonization of one or more of the fungal pathogens in tissues and organs of an infected subject, such as lung or brain tissue.
  • the invention provides a method of providing immunity (or immune protection) to a subject in need who has an infection or who is at risk of having an infection by one or more, or two or more, different fungal types, including Pneumocystis, Aspergillus, Candida and Cryptococcus fungal organisms, by administering to the subject an isolated antiserum, or monoclonal or polyclonal antibodies isolated and purified from a
  • the Kex peptide is an antigenically stable active site peptide sequence of a Kexin protein as described herein.
  • the isolated antiserum is generated against a Pneumocystis Kex peptide.
  • the isolated antiserum is generated against an Aspergillus Kex peptide.
  • the isolated antiserum is generated against a Candida Kex peptide.
  • the isolated antiserum is generated against a Cryptococcus Kex peptide.
  • the antiserum is generated in (and isolated from) one organism or subject and administered to another organism or subject who has or who is at risk of having an infection by one or more of the fungal organisms described herein.
  • the immunity provided by the practice of the method is humoral immune protection (antibody protection) against one or more of the fungal organisms described herein, in particular, against the Kex protein or Kex peptide of one or more of the described fungal organisms.
  • the immunity provided by the practice of the method is an acquired or passive humoral immune protection (antibody protection) provided to a recipient subject against one or more of the fungal organisms described herein, in particular, against the Kex protein or Kex peptide of one or more of the described fungal organisms.
  • the isolated antiserum is administered in a pharmaceutically acceptable composition.
  • the invention provides a method of treating or preventing infection caused by one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms, in which the method involves administering to a subject who is infected or who is at risk of infection with an isolated antiserum containing antibodies that are generated against and target the Kex protein or a Kexl peptide as described herein, or naturally occurring anti- Kex protein or peptide monoclonal or polyclonal antibodies isolated and purified from a nonimmunized subject, wherein the Kex protein or a Kex peptide thereof is from at least one of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms.
  • the Kex peptide is an antigenically stable active site peptide sequence of the Kexin protein, which comprises conserved amino acids among several different fungal organisms, namely, Pneumocystis hominis, Aspergillus fumigates, Candida albicans and Cryptococcus neoformans.
  • the isolated antiserum is administered in a pharmaceutically acceptable composition.
  • the invention provides a method of treating or preventing a fungal induced pulmonary disease or symptoms thereof in a subject, the method involving administering to a subject in need thereof an isolated anti -Kexin protein or peptide antibody or an antigen-binding fragment thereof generated against a Kex peptide from one type of fungal organism, e.g., Pneumocystis, or an isolated antiserum comprising such an antibody or an antigen binding fragment thereof, which antibody, antigen binding fragment thereof, or isolated antiserum immunizes the subject against pulmonary disease caused by other fungal types, for example, Aspergillus, Candida and Cryptococcus fungal pathogens, and more specifically, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans fungal pathogens.
  • an isolated anti -Kexin protein or peptide antibody or an antigen-binding fragment thereof generated against a Kex peptide from one type of fungal organism e.g., Pneu
  • the method involves administering anti-fungal Kexin protein or peptide monoclonal or polyclonal antibodies isolated and purified from a nonimmunized subject as described herein.
  • the isolated antiserum or the isolated and purified naturally occurring antibodies are administered in a pharmaceutically acceptable composition.
  • an antiserum isolated from a subject immunized with an approximately 90 amino acid peptide segment of the subtilisin-like serine proteinase Kexin of Pneumocystis (Pneumocystis KEX1 (Accession no. EU918304.1), namely, a Kex peptide, comprises antibodies that also recognized Kex peptides derived from different fungal pathogens, such as Pneumocystis, Aspergillus, Candida and Cryptococcus fungal pathogens, and more specifically, in the Pneumocystis hominis, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans fungal species.
  • antiserum isolated antiserum
  • the antibodies contained therein can serve as a therapeutic or protective treatment not only against infection or disease associated with Pneumocystis infection, but also against infection or disease associated with infection by one or more of the Aspergillus, Candida and Cryptococcus fungal pathogens in a subject immunized with or administered the antiserum.
  • the antiserum treats or protects against pulmonary disease, such as poor pulmonary function, COPD, or pneumonia.
  • the methods and compositions described herein are advantageously useful for providing high levels of antibodies (and increased antibody titers) directed against an antigenic region of the Kexin protein in the Pneumocystis, Aspergillus, Candida and Cryptococcus fungal pathogens to a subject in need thereof.
  • the methods are useful for the treatment or protection of cigarette smokers who are at risk for COPD and in patients with COPD, so as to ameliorate airway obstruction and to treat or prevent more severe airway obstruction and progressive COPD in these individuals.
  • compositions and methods described herein are advantageously useful for providing high levels of cross-reactive antibodies (and increased antibody titers) directed against Kexl peptides of the Pneumocystis, Aspergillus, Candida and Cryptococcus fungal pathogens.
  • the methods are useful for the treatment or protection of a subject in need from colonization of tissue, e.g., lung tissue, with the Pneumocystis, Aspergillus, Candida and Cryptococcus Pneumocystis fungal pathogens.
  • the fungal pathogen is
  • the antiserum is an isolated antiserum or a purified antiserum. In an embodiment,
  • antibodies from the antiserum may be purified by methods known and practiced in the art, and the purified antibodies (in a pharmaceutically acceptable composition) may be provided to a recipient subject.
  • the invention provides a kit containing the isolated antiserum containing antibodies or antigen-binding fragments thereof generated against a Kex peptide according to any aspect delineated herein.
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • the kit contains antibodies or antigen binding fragments thereof purified from an anti-fungal Kex peptide antiserum obtained or isolated as described herein and in a pharmaceutically acceptable composition.
  • the subject is human.
  • the subject is infected with or is at risk of infection with a fungal organism selected from the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • the subject has or is at risk of having a pulmonary disease, e.g., COPD.
  • the fungal infection is neutralized in the subject.
  • tissue colonization by a fungal pathogen is reduced, diminished, or alleviated in a subject.
  • the antibody or antigen-binding fragment thereof is obtained from blood, plasma, or serum.
  • a method of cross-protecting a subject against infection and/or disease by different fungal pathogens involves administering to a subject in need thereof an isolated antiserum produced against a peptide immunogen derived from the Kexin protein of a Pneumocystis fungal pathogen in an amount effective for the subject to acquire cross-protective immunity (e.g., humoral immunity) against infection and/or disease associated with one or more of an Aspergillus, Candida, or Cryptococcus fungal pathogen.
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • a method of treating a subject having a disease or dysfunction associated with infection by different fungal pathogens involves administering to a subject in need thereof a peptide immunogen derived from the Kexin protein of a Pneumocystis fungal pathogen or an isolated antiserum produced against a peptide immunogen derived from the Kexin protein of a Pneumocystis fungal pathogen in an amount effective to treat the subject for the disease or dysfunction associated with infection by one or more of an Aspergillus, Candida, or Cryptococcus fungal pathogen.
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • the antiserum is generated against a Kex peptide of Pneumocystis hominis or Pneumocystis jirovecii.
  • the cross-protection or treatment is against infection and/or disease or dysfunction associated with one or more of Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans.
  • the subject is cross-protected against or treated for a disease or dysfunction selected from pulmonary disease, chronic obstructive pulmonary disease (COPD), poor pulmonary function, or a symptom thereof.
  • COPD chronic obstructive pulmonary disease
  • the antiserum is generated in the subject against the 90- amino acid Kex peptide derived from the Pneumocystis hominis or Pneumocystis jirovecii fungal pathogen. (FIG. 3).
  • the antiserum is an isolated antiserum which allows a recipient subject to acquire immunity to multiple fungal pathogens as described herein.
  • a method of cross-protecting a subject against infection and/or disease by different fungal pathogens involves administering to a subject in need thereof an isolated antiserum produced against a peptide immunogen derived from the Kexin protein of a fungal pathogen selected from one or more of
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • a method of treating a subject having a disease, dysfunction, or symptoms thereof associated with infection by different fungal pathogens involves administering to a subject in need thereof an isolated antiserum produced against a peptide immunogen derived from the Kexin protein of a fungal pathogen selected from one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus in an amount effective for the subject to acquire immunity against and to treat a disease, dysfunction, or symptoms thereof associated with infection by at least two of the
  • the peptide immunogen is a Kexl peptide derived from
  • the Pneumocystis Kexl peptide is a 90-amino acid peptide (FIG. 3) and is encoded by a polynucleotide contained in an expression vector.
  • the peptide immunogen is a Kexl peptide derived from Candida albicans.
  • the Candida Kexl peptide is an 88-amino acid peptide (FIG. 3) and is encoded by a polynucleotide contained in an expression vector.
  • the peptide immunogen is a Kexl peptide derived from Aspergillus fumigatus.
  • the Aspergillus Kexl peptide is an 88-amino acid peptide (FIG. 3) and is encoded by a polynucleotide contained in an expression vector.
  • the peptide immunogen is a Kexl peptide derived from Cryptococcus neoformans.
  • the Cryptococcus Kexl peptide is a 117- amino acid peptide (FIG. 3) and is encoded by a polynucleotide contained in an expression vector. (FIG. 3).
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • a method of treating or preventing an infection involves administering to a subject who is infected or who is at risk of infection by two or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens an isolated antiserum generated in a different subject (a donor subject) who has mounted an antibody-mediated immune response against one of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • the antiserum comprises one or more antibodies or antigen-binding fragments thereof that target and bind to a Kex peptide of a Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogen, in particular, two or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • a method of treating or preventing an infection in which the method involves administering to a subject infected or at risk of infection by
  • Pneumocystis Aspergillus, Candida, or Cryptococcus fungal pathogens an antiserum isolated from a donor subject who has generated an immune response to a Kex peptide of a
  • a method of treating or preventing an infection in which the method involves administering to a subject infected or at risk of infection by
  • Pneumocystis Aspergillus, Candida, or Cryptococcus fungal pathogens an antiserum isolated from a donor subject who has generated an immune response to a Kex peptide of an
  • a method of treating or preventing an infection in which the method involves administering to a subject infected or at risk of infection by
  • Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens an antiserum isolated from a donor subject who has generated an immune response to a Kex peptide of a Candida fungal pathogen.
  • a method of treating or preventing an infection in which the method involves administering to a subject infected or at risk of infection by
  • Pneumocystis Aspergillus, Candida, or Cryptococcus fungal pathogens an antiserum isolated from a donor subject who has generated an immune response to a Kex peptide of a
  • the antiserum is an isolated or purified antiserum. In an embodiment of any of the above aspects, the antiserum has neutralizing activity against a fungal pathogen or prevents colonization of a fungal pathogen in the subject. In an embodiment of any of the above aspects, the donor and recipient subjects are mammals, particularly, human beings. In another embodiment of the above aspects, the recipient subject has or is at risk of having an infection or disease caused by more than one, e.g., two or more, fungal pathogen(s) selected from Pneumocystis, Aspergillus, Candida, or Cryptococcus. In an embodiment of any of the above aspects, the subject receiving the isolated or purified antiserum is immunocompromised.
  • the subject receiving the isolated or purified antiserum is cross-protected against or treated for a disease or dysfunction or symptom thereof selected from pulmonary disease, chronic obstructive pulmonary disease (COPD), poor pulmonary function, or pneumonia, caused by or associated with infection by a fungal pathogen as described herein.
  • a disease or dysfunction or symptom thereof selected from pulmonary disease, chronic obstructive pulmonary disease (COPD), poor pulmonary function, or pneumonia, caused by or associated with infection by a fungal pathogen as described herein.
  • COPD chronic obstructive pulmonary disease
  • the invention encompasses a kit for detecting, qualifying, or quantifying the levels of, antibodies directed against the Kex protein derived from
  • kits comprising a solid substrate having attached thereto a Kex peptide derived from one or more, two or more, three or more, or each of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • the kit further comprises a labeled detection molecule for detecting and measuring the level of antibodies that bind to the Kex peptides on the substrate.
  • the kit is used for performing an immunoassay, which may be an enzyme linked immunosorbent assay (ELISA).
  • the kit also contains a positive and/or a negative control for detecting, qualifying, or quantifying antibody levels.
  • the invention encompasses a composition comprising a solid substrate and a plurality of Kex peptides derived from one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens immobilized on the substrate.
  • Cryptococcus fungal pathogens is immobilized at a different, indexable, location on the substrate.
  • a mixture of Kex peptides from two or more, three or more, or all four of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens is immobilized at a different, indexible, location on the substrate.
  • the binding of anti-Kex peptide antibodies from a sample obtained from a subject can be measured or detected by measuring or detecting complexes of the anti-Kex peptide antibodies bound to the Kex peptides localized on the substrate.
  • the composition is contained in a kit for performing an immunoassay, which may be an enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • the invention provides a method of diagnosing a subject as being at risk of infection by one or more fungal pathogens and/or fungal-associated disease or symptoms thereof, in which the method comprises: contacting a Kexin (Kex) protein or peptide derived from at least two of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens with a biological sample of the subject to detect antibodies or antigen binding fragments thereof in the sample that specifically bind to the Kex protein or peptide; detecting the binding levels of the anti-Kex protein or peptide antibodies or antigen binding fragments thereof in the sample to the Kex protein or peptide relative to control binding levels; and diagnosing the subject as being at risk of infection or disease by the fungal pathogens when low or negligible levels of the Kex protein or peptide binding
  • normal human sera with undetectable absorbance at OD450 i.e., equal to or less than the OD450 absorbance measured for dilution buffer alone
  • KEX- ELISA normal human sera with undetectable absorbance at OD450 (i.e., equal to or less than the OD450 absorbance measured for dilution buffer alone) in KEX- ELISA at a dilution of 1 : 100 is used as a negative control.
  • negative to low titer range is between about 1 to about 3200 RET, moderate titer levels are about 3200 to about 12,800 and high titer levels are greater than (>) 12,800 RET.
  • the invention provides a method of stratifying a subject into a patient population that is at risk or that is not at risk of being infected by one or more fungal pathogens and/or disease associated therewith, in which the method comprises: contacting a Kexin (Kex) protein or peptide derived from one or more fungal pathogens selected from Pneumocystis, Aspergillus, Candida, or Cryptococcus with a biological sample of the subject to detect antibodies or antigen binding fragments thereof in the sample that specifically bind to the Kex protein or peptide; detecting and measuring levels of antibodies or antigen binding fragments thereof in the subject's sample that specifically bind to one or more of the fungal- derived Kex protein or peptide; and stratifying the subject into (i) a patient population that is at risk of having infection and/or disease associated with exposure to one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens when low or negligible levels
  • moderate to high levels or titers of specific mti-Pneumocystis Kex protein or peptide antibodies or antigen binding fragments thereof in the subject's sample provide protection to the subject from infection and/or disease caused by one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • negative to low antibody titers range from about 1 to about 3200 RET
  • moderate levels range from about 3200 to about 12,800 reciprocal endpoint antibody titers (RET)
  • high levels are greater than about 12,800 RET, based on the distribution of anti- Pneumocystis KEXl RET in human samples.
  • the Kex protein or peptide is directly or indirectly coupled to a detectable substance.
  • the anti-Kex protein or peptide antibody or an antigen-binding fragment thereof bound to the Kex protein or peptide is detected by a secondary antibody or an antigen binding fragment thereof that is directly or indirectly coupled to a detectable substance selected from one or more of an enzyme, prosthetic group, fluorescent material, luminescent material, bioluminescent material, radioactive material, positron emitting metal, or a nonradioactive paramagnetic metal ion.
  • the fungal-derived Kex protein or peptide is attached to or immobilized on a solid support.
  • the binding of the anti-Kex protein or peptide antibodies or antigen-binding fragments thereof in the subject's sample to the fungal-derived Kex protein or peptide is detected by an immunoassay.
  • a subject who has low, no, or negligible levels of anti -fungal-derived Kex protein or peptide antibodies or antigen binding fragments thereof in her/his sample is treated with an antibody or an antigen binding fragment thereof directed against one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens so as to provide therapeutic protection to the subject against infection and/or disease caused by the one or more fungal pathogens.
  • the invention provides a method of treating or protecting an immunosuppressed patient against infection by one or more fungal pathogens and/or disease associated with infection by the fungal pathogens, in which the method comprises: administering to a subject who is to receive, is receiving, or has received an immune suppressive drug or medication, a peptide immunogen derived from the Kexin protein of a fungal pathogen, an isolated antiserum comprising antibodies produced against the peptide immunogen, or isolated and purified antibodies produced against the peptide immunogen, wherein the fungal pathogen is selected from one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus in an amount effective for the patient to acquire cross-protective immunity against infection and/or disease associated with two or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • the patient is a pre-transplant patient or a post-transplant patient.
  • the patient is to receive or has received an organ transplant selected from a kidney, liver, heart, bone marrow, pancreas, lung, gall bladder, or bladder organ transplant.
  • the patient suffers from rheumatoid arthritis or psoriasis.
  • the patient is receiving or has received one or more organ transplants selected from a kidney, liver, heart, bone marrow, pancreas, lung, gall bladder, or bladder organ transplant.
  • the patient suffers from rheumatoid arthritis or psoriasis.
  • the patient is receiving or has received one or more
  • immunosuppressive drugs selected from corticosteroids, calcineurin inhibitors, mTOR inhibitors, inosine monophosphate dehydrogenase (IMDH) inhibitors, a biologic or a monoclonal antibody or an antigen binding fragments thereof.
  • Kexl or “Kexin” or “KEX” protein is meant a polypeptide or peptide fragment thereof having at least about 85% or greater amino acid identity to the amino acid sequence provided at GenBank Accession No. EU918304.1, at NCBI Accession No. XM_746441.1, at GenBank Accession No. AF022372.1, or at NCBI Accession No. XM_572303.1 and having biological activity as a serine protease.
  • the Kex peptide is an antigenically stable active site peptide sequence.
  • Kex (KexB endoprotease) polypeptide sequence of Aspergillus fumigatus Af293 having NCBI Accession No. XM_746441.1 is provided below:
  • agent is meant a peptide, nucleic acid molecule, or small compound.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • alteration is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein.
  • an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels.
  • antibody refers to an immunoglobulin molecule which specifically binds with an antigen. Methods of preparing antibodies are well known to those of ordinary skill in the science of immunology. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Tetramers may be naturally occurring or reconstructed from single chain antibodies or antibody fragments. Antibodies also include dimers that may be naturally occurring or constructed from single chain antibodies or antibody fragments.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab') 2 , as well as single chain antibodies (scFv), humanized antibodies, and human antibodies (Harlow et al, 1999, In: Using
  • Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al, 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al, 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
  • antibody fragment refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody.
  • antibody fragments include, but are not limited to, Fab, Fab', F(ab') 2, and Fv fragments, linear antibodies, scFv antibodies, single-domain antibodies, such as camelid antibodies
  • the antibody fragment also includes a human antibody or a humanized antibody or a portion of a human antibody or a humanized antibody.
  • Antibodies can be made by any of the methods known in the art utilizing a polypeptide (e.g., a Kexin polypeptide), or immunogenic peptide fragments thereof, as an immunogen.
  • a polypeptide e.g., a Kexin polypeptide
  • immunogenic peptide fragments thereof an immunogen.
  • One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production.
  • the immunogen facilitates the presentation of the immunogenic fragments on the cell surface.
  • Immunization of a suitable host can be carried out in several ways. Nucleic acid sequences encoding a polypeptide of the invention, or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in rum express the receptor on the cell surface generating an immunogenic response in the host.
  • nucleic acid sequences encoding the polypeptide, or immunogenic fragments thereof
  • antibodies against the polypeptide may, if desired, be derived from an antibody phage display library.
  • a bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human antibody genes, to display human antibody proteins.
  • Phage display is the process by which the phage is made to 'display' the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.
  • Antibodies made by any method known in the art can then be purified from the host.
  • Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.
  • Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art.
  • the hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid.
  • the method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane).
  • a suitable composition e.g., Pristane
  • anti-Kexin antibody By “anti-Kexin antibody,” “anti-Kex antibody,” or “anti-Kexl antibody” is meant an antibody or an antigen binding fragment thereof that selectively binds a Kexin polypeptide or a peptide fragment thereof, including, for example, a Kexl peptide fragment of a fungal pathogen, such as Candida albicans, Pneumocystis hominis, Pneumocystis jirovecii (aka carinii), Aspergillus fumigatus and Cryptococcus neoformans as described herein.
  • anti-Kexin antibody or anti-Kexl antibody specifically binds a binding site of a Kexin protein or peptide.
  • the anti-Kexin antibody or anti-Kexl antibody specifically binds a binding site of a Kexin protein or peptide of a fungal pathogen, for example, one or more of a Candida, Pneumocystis, Aspergillus and/or Cryptococcus fungal pathogen.
  • a Kexin protein or peptide of a fungal pathogen for example, one or more of a Candida, Pneumocystis, Aspergillus and/or Cryptococcus fungal pathogen.
  • the "anti-Kexin antibody” or “anti-Kexl antibody” has at least about 85% or greater amino acid identity to a Kexl peptide amino acid sequence provided below.
  • an “antiserum” refers to blood serum that contains one or more antibodies directed against a specific antigen.
  • Antiserum containing antibodies may be obtained from the blood or serum of an animal (a mammal), including a human, that has been immunized or inoculated with an immunogen (or an antigen material) either by injection, typically into the bloodstream or tissues, or by infection.
  • the animal (a mammal), including a human may be immunized or inoculated with the blood or serum of an organism or individual whose immune system has been stimulated to generate an immune response (e.g., antibody production) by infection or natural contact with an antigenic material or
  • an antiserum contains anti-Kex peptide antibodies, e.g., polyclonal antibodies or populations of monoclonal antibodies, generated or produced by an immunized, inoculated, or exposed donor subject against a Kex peptide immunogen derived from a fungal pathogen, e.g., Pneumocystis (e.g., Pneumocystis jirovecii).
  • a fungal pathogen e.g., Pneumocystis (e.g., Pneumocystis jirovecii).
  • Such antiserum, isolated (and/or purified) from the donor subject is used to immunize (i.e., administer to) another (unrelated) subject so as to provide immunity (acquired immunity) against infection or disease caused by or associated not only with the Pneumocystis pathogen as original source of the immunogen, but also with other fungal pathogens that have a Kex peptide that is also targeted and recognized by the antibodies in the antiserum.
  • the fungal pathogens include Pneumocystis species (spp.) and one or more of Candida spp. or Candida albicans,
  • a subject who receives the antiserum i.e., antibodies in the antiserum, is treated or protected against infection and/or disease caused by more than one fungal pathogen.
  • antiserum-derived immunoprotection against multiple fungal pathogens constitutes an acquired or passive immunity obtained by the recipient subject and imparted from the donor subject's isolated antiserum.
  • blood serum is the amber-colored, protein-rich liquid component of blood that separates from the clot when blood coagulates.
  • the serum component containing one or more antibodies is termed "antiserum.”
  • the antiserum is an isolated antiserum, e.g., isolated from a donor subject.
  • an isolated antiserum may be processed by methods used by one skilled in the art, such as dilution, concentration (e.g., via filtration or centrifugation or both), chromatography, purification to remove ions or extraneous protein, and the like, prior to its use as a treatment or protective therapeutic as described herein.
  • an isolated antiserum may be further purified after isolation.
  • an isolated antiserum is not further processed or purified.
  • antibodies, or antigen-binding fragments thereof, contained in an isolated antiserum may be further isolated by methods practiced by those having skill in the art, such as, without limitation, by affinity chromatography, size exclusion chromatography, immunoprecipitation, dialysis, HPLC chromatography, etc.
  • biological sample any liquid, cell, or tissue obtained from a subject.
  • the biological sample is blood, serum, plasma, cerebrospinal fluid, bronchoalveolar lavage, sputum, tears, saliva, urine, semen, feces, etc.
  • Detect refers to identifying the presence, absence or amount of the analyte that is detected or that is to be detected.
  • disease is meant any condition, dysfunction, or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • the disease is a pulmonary (lung) disease or a brain disease, e.g., meningitis.
  • pulmonary diseases include Chronic Obstructive Pulmonary Disease (COPD), which is a progressive lung disease that includes emphysema, chronic bronchitis, severe refractory (non-reversible) asthma, and some forms of bronchiectasis.
  • COPD Chronic Obstructive Pulmonary Disease
  • an immunologically effective amount of an isolated antiserum of the invention is an amount required to treat a fungal infection or disease associated with one or more of the fungal pathogens described herein.
  • an effective amount of an isolated antiserum may be determined by measuring the amount or titer of antibodies directed against the desired immunogen present in the serum by methods known and practiced in the art.
  • the range of typical dosages for passive immunotherapy i.e., the administration of antiserum containing antibodies
  • treatment efficacy is typically conducted, as individual patients respond differently to therapies. Adjustment of the dosage may be modified as needed.
  • Treatment regimens can be determined by methods known and practiced by those having skill in the art.
  • fragment is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.
  • isolated refers to material that is free to varying degrees from components which normally accompany it as found in its native state or environment.
  • Isolate denotes a degree of separation from original source or surroundings.
  • Purify denotes a degree of separation that is higher than isolation.
  • biologically pure protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high-performance liquid chromatography. The term “purified” can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.
  • isolated polynucleotide is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene.
  • the term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences.
  • the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
  • an “isolated polypeptide” or “isolated peptide” is meant a polypeptide or peptide that has been separated from components that naturally accompany it.
  • the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention.
  • An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • obtaining as in “obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.
  • reference is meant a standard or control condition.
  • a “reference sequence” is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.
  • cross-reactive binding includes specific binding (e.g., by an antibody or an antigen binding fragment thereof) to an original polypeptide or peptide antigen/immunogen as well as binding to a polypeptide or peptide other than the original antigen/immunogen.
  • Nucleic acid molecules useful in generating a recombinant immunogen or a vaccine include any nucleic acid molecule that encodes a polypeptide or a peptide fragment thereof, such as a Kex polypeptide or Kex peptide described herein. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity to an endogenous sequence. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules may include any nucleic acid molecule that encodes a polypeptide or a peptide fragment thereof.
  • Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.
  • hybridize is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency.
  • complementary polynucleotide sequences e.g., a gene described herein
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • a reference amino acid sequence for example, any one of the amino acid sequences described herein
  • nucleic acid sequence for example, any one of the nucleic acid sequences described herein.
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine;
  • BLAST program may be used, with a probability score between e "3 and e -100 indicating a closely related sequence.
  • subject is meant a mammal, including, but not limited to, a human or non- human mammal, such as a non-human primate, or a murine, bovine, equine, canine, ovine, or feline mammal.
  • the subject is a human.
  • a subject is a human patient who is undergoing treatment for infection or disease caused by one or more pathogenic fungi, such as Pneumocystis, Aspergillus, Candida, or Cryptococcus .
  • a subject is a human patient who is at risk of infection (e.g., opportunistic infection) or disease caused by one or more pathogenic fungi, such as Pneumocystis, Aspergillus, Candida, or Cryptococcus.
  • a subject is a mammalian (e.g., a human; a non-human primate) donor subject from whom antiserum containing anti-fungal Kex peptide antibodies is obtained or isolated.
  • a subject is a mammalian (e.g., a human; a non-human primate) recipient subject who receives an isolated antiserum and acquires protective immunity (and treatment) against multiple fungal pathogens.
  • opportunistic infection is meant an infection caused by pathogens such as fungal pathogens, bacteria, viruses, protozoa, or parasites that take advantage of an opportunity to infect a subject (host) that is not normally available, for example, a host having a weakened immune system, an immunocompromised host, a host with altered microbiota or microflora, or a host having protective integumentary barriers that have been damaged or breached.
  • an opportunistic infection is caused by one or more fungal pathogens as described herein.
  • PS-15 refers to a dihydrofolate reductase inhibitor having the following structure:
  • QS-21 refers to an adjuvant having the following structure:
  • Ranges provided herein are understood to be shorthand for all the values within the range.
  • a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.
  • the terms "treat,” treating,” “treatment,” and the like refer to reducing, abating, diminishing, or ameliorating a disease, disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disease, disorder and/or symptoms associated therewith does not require that the disease, disorder, condition or symptoms associated therewith be completely eliminated.
  • a therapeutic that "prevents" a disorder or condition refers to a compound or material that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample.
  • a preventive therapeutic is an antibody or an antigen binding fragment thereof.
  • a preventive therapeutic is an isolated antiserum containing anti-Kex peptide antibodies or antigen binding fragments thereof as described herein.
  • the term "about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • FIGS. 1A and IB present a multiple amino acid sequence alignments
  • FIG. 1 A provides a multiple sequence alignment comparing a 90-amino acid region of the subtilisin-like serine protease Kexin (KEX1 or KEX) derived from Pneumocystis jirovecii (P. jirovecii KEX1); Pneumocystis macaque (P. macaque KEX1); Aspergillus fumigatus (Aspergillus KEX); Cryptococcus neoformans (Cryptococcus KEX); and Candida albicans ⁇ Candida KEX).
  • KEX1 or KEX subtilisin-like serine protease Kexin
  • FIG. 1A depicts phylogenic genetic tree data comparing the KEX peptides of Pneumocystis and other fungal pathogens reflected in FIG. 1A.
  • FIG. 2 presents a multiple sequence alignment analysis of Kexin (KEXl, KEX) peptide identity and similarity.
  • FIG. 3 illustrates the sequences of KEXl peptides from Pneumocystis, Aspergillus, Candida, and Cryptococcus contained in expression constructs expressed in ?. coli (i.e., recombinant fungal KEXl homologs as expressed in E. coli).
  • the highlighted regions are the sequences expressed by the expression constructs.
  • Pneumocystis KEXl and homologous regions are shown.
  • FIG. 3 (1.), a Pneumocystis Kexl conserved 90 amino acid sequence used in an expression construct is shown; in FIG. 3 (2.), a 90 amino acid macaque Pneumocystis Kexl conserved sequence used in an expression construct is shown; in FIG. 3 (3.), a conserved 90 amino acid Aspergillus Kex sequence is shown; and an 88 amino acid Aspergillus Kexl sequence used in an expression construct (designated
  • FIG. 3 a conserved 90 amino acid Candida Kex sequence is show; and an 88 amino acid Candida Kex sequence used in an expression construct
  • Cryptococcus Kex sequence is shown; and a Cryptococcus Kex expression construct (designated CN_Kex.DG117) containing 117 amino acid residues is shown.
  • CN_Kex.DG117 construct shown in FIG. 3 contained a polynucleotide encoding a 117- amino acid sequence, which included an additional 28 amino acids downstream as shown (DHIHTTDVGKDKCSHSHGGTSAAAPLAV).
  • FIGS 4A-4D present Western blots which show that the Candida Kex recombinant protein is recognized by (immunologically cross-reactive with) sera (antisera containing antibodies) from monkeys (macaques) immunized with Pneumocystis KEXl (P. macaque, Accession No. EU918304.1).
  • An 88-amino acid Candida KEX peptide (FIGS. 1A and 3) containing an N-terminal tobacco etch virus (TEV) cleavage site and a maltose binding protein (MBP) tag ⁇ Candida KEX-MBP) was recombinantly expressed and affinity purified. (See, Example 2).
  • Blots were then incubated with goat anti-monkey IgG (H+L)-HRP secondary antibody (1 : 10,000) in blocking buffer for 1 hour at room temperature and were visualized with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher). As a negative control, parallel blots were probed with secondary antibody only, shown in FIG. 4D.
  • FIGS. 5A-5D present Western blots which demonstrate that the Aspergillus Kex recombinant protein is recognized by sera from monkeys (macaques) immunized with
  • Pneumocystis KEX 1 P. macaque, Accession No EU918304.1. Accordingly, the Aspergillus Kex recombinant protein is immunologically cross-reactive with Pneumocystis Kexl antibodies in the antiserum.
  • An 88-amino acid long Aspergillus KEX peptide (FIGS. 1A and 3) containing an N-terminal TEV cleavage side and maltose binding protein (MBP) tag (Aspergillus KEX-MBP) was recombinantly expressed and affinity purified. (See, Example 2).
  • the purified recombinant protein was then incubated with TEV protease to cleave the N- terminal MBP affinity tag (Aspergillus KEX-MBP + TEV). Recombinant proteins were resolved by 15% SDS-PAGE and immunoblotting was performed as described in FIGS. 4A- D.
  • membranes were blotted with high titer sera from vaccinated monkey 17911 (dilution 1 :500), shown in FIG. 5A, or with low titer sera from unvaccinated monkey 8015 (dilution 1 :500), shown in FIG.
  • FIG. 5B For 2 hours at room temperature.
  • the presence of MBP tagged proteins was confirmed by immunoblotting with mouse anti-MBP antibody, shown in FIG. 5C.
  • FIG. 5D As a negative control, parallel blots were probed with secondary antibody only, as shown in FIG. 5D.
  • FIGS. 6A and 6B present Western blots probed with animal sera as described herein.
  • FIG. 6 A shows Western blots showing that Aspergillus KEX is recognized by the immune system during Aspergillus infection in animals in vivo and at low levels in uninfected animals. The animals produce ti-Aspergillus antibodies during Aspergillus infection.
  • Recombinant Aspergillus KEX-MBP and TEV-cleaved Aspergillus KEX ⁇ Aspergillus KEX- MBP + TEV) proteins were resolved by 15% SDS-PAGE and immunoblotting was performed as described for FIGS. 4A-D above.
  • Aspergillus KEX peptide behaved as an immunogen and was detected by an animal's immune system during a natural infection with Aspergillus
  • membranes were blotted with sera obtained from an Aspergillus- infected mouse (Top Panel), or with sera from an uninfected mouse (Bottom Panel) at the dilutions indicated above the lanes at the top of the blots, i.e., 1 to 50 (1 :50); 1 to 100 (1 : 100); and 1 to 200 (1 :200).
  • FIG. 6B presents a Western blot showing that antisera generated during Aspergillus infection is immunologically cross-reactive with recombinant KEX proteins of Cryptococcus , Candida and Pneumocystis. Recombinant KEX-MBP proteins and TEV-cleaved KEX proteins were resolved by 15% SDS-PAGE and immunoblotting was performed as described for FIGS. 4A-D above.
  • membranes were blotted with sera from an Aspergillus-mfected mouse at the dilution of 1 :500. The blots were then incubated with goat anti-mouse IgG (H+L)-HRP secondary antibody (1 : 10,000) (ThermoFisher) and were visualized with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher).
  • Recombinant proteins on the blot were expressed as fusions with maltose binding protein (MBP) cleaved with TEV protease to release fungal peptides, namely, Cryptococcus KEX, Aspergillus KEX, Candida KEX, and Pneumocystis KEXl.
  • MBP maltose binding protein
  • TEV protease to release fungal peptides
  • FIGS. 7A and 7B depict diagrams of template plates for performing an ELISA analysis as described in Example 6.
  • FIG. 7A depicts an ELISA analysis template in which vertical dilutions are made using 10 test samples and 1 control on the microtiter plate.
  • FIG. 7B depicts an ELISA analysis template in which horizontal dilutions are made using 6 test samples and 1 control on the plate.
  • FIG. 8 shows a graph of a titer curve based on data obtained from the ELISA analysis performed as described in Example 6 and shown in the table therein. Reciprocal endpoint titers are estimated as follows: For sample #951, the endpoint titer value is 1200; for sample #962, the endpoint titer value is 350; and for sample #966, the endpoint titer value is 350.
  • FIG. 9 presents graphs of the data observed in standard KEXl ELISA analyses of sera obtained from a cohort or pediatric severe asthmatics and a cohort of pediatric cystic fibrosis patients.
  • the invention generally provides methods for treating or preventing infection by one or more fungal pathogens, or disease associated with infection by more than one fungal pathogen, in which the fungal organisms are of different etiologies.
  • the invention embraces an immunogenic composition that elicits a potent immune response in a subject following administration of the composition and the production of antiserum in the subject that contains one or more antibodies or antigen binding fragments thereof that reacts not only with the immunizing antigen, but also with a similar, but nonidentical, antigen found in other, distinct fungal pathogens.
  • One benefit of the described methods is the provision of treatment or prevention of infection by and disease associated with several different fungal pathogens using only one therapeutic agent, i.e., an antiserum (isolated antiserum), that cross-protects against multiple fungal organisms and treats or prevents diseases and symptoms thereof, for example, pulmonary disease and poor pulmonary performance, associated with infection (and colonization) by the different fungal pathogens, e.g., at least two or more fungal pathogens.
  • the different fungal pathogens include Pneumocystis, Aspergillus, Candida, and Cryptococcus, particularly, at least two or more thereof.
  • an antiserum produced in a subject immunized with a Kex peptide derived from Pneumocystis ⁇ Pneumocystis jirovecii) contains antibodies that also specifically react with a Kex peptide derived from one or more of Aspergillus ⁇ Aspergillus fumigatus), Candida ⁇ Candida albicans), or Cryptococcus ⁇ Cryptococcus neoformans).
  • the antiserum is cross-protective (e.g., cross-reactive with Kex peptides in multiple fungal types) and affords treatment and/or protection against infection and/or disease associated with multiple fungal organisms when provided to another (e.g., unrelated) subject in need thereof.
  • the antiserum is an isolated antiserum.
  • the isolated antiserum is administered in a pharmaceutically acceptable composition.
  • the methods and compositions described herein offer economic, medical and practical benefits in the treatment and prevention of fungal infection and disease, such as pulmonary disease, or types of brain infections, associated with infection and colonization by different types of fungal pathogens.
  • the immunogen or immunogenic composition comprising a Pneumocystis-derived Kex peptide described herein generated specific antiserum against Pneumocystis Kex peptide; this antiserum was surprisingly found to react specifically against Kex peptides of one or more other fungal pathogens, e.g., one or more of Aspergillus (Aspergillus fumigatus), Candida (Candida albicans), or Cryptococcus
  • the described methods involve the use of antiserum (e.g., an isolated antiserum) that can provide to a subject to whom the antiserum is administered immunity against infection and disease associated with more than one fungal organism.
  • antiserum e.g., an isolated antiserum
  • the recipient subject is infected or is at risk of becoming infected with more than one disease-causing fungal pathogen, namely, Pneumocystis, Aspergillus, Candida and Cryptococcus, and, in particular, Pneumocystis hominis or jirovecii, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans.
  • the antiserum is an isolated antiserum.
  • the isolated antiserum is in, or is
  • compositions provided herein can be used to treat or prevent infection and/or associated disease caused by the fungal pathogens Pneumocystis,
  • the methods and compositions provided herein can provide immune protection in a subject against infection and disease caused by at least one, and particularly more than one, of these fungal organisms.
  • the methods and compositions provided herein can immunize a recipient subject against infection by at least one and particularly by more than one of these fungal organisms.
  • isolated antiserum containing one or more antibodies generated against a Kex peptide of Pneumocystis, or of Aspergillus, Candida, or Cryptococcus fungal organisms can be administered therapeutically and/or prophylactically to provide immunity against other pathogenic fungal organisms that express a Kex protein or peptide antigen.
  • the methods include administering an immunologically effective amount of an isolated antiserum, or immune serum or immune plasma, described herein to an individual, alone, or in a physiologically acceptable carrier, excipient, or diluent.
  • Cryptococcus more specifically, Pneumocystis, Aspergillus, Candida, or Cryptococcus fungi, or antiserum obtained or isolated from an immune survivor of infection by one of these fungal pathogens, is used to treat or prevent the infection by another or different type or species of fungal pathogen as described herein.
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • the present invention provides methods of treating or preventing an infection by one or more fungal pathogens (e.g., one or more of Pneumocystis , Aspergillus, Candida, or Cryptococcus, more specifically, Pneumocystis hominis or jirovecii, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans fungi), and/or diseases, disorders, or symptoms thereof, which comprise administering a therapeutically effective amount of an isolated antiserum as described herein to a recipient subject (e.g., a mammal such as a human patient) in need thereof.
  • a recipient subject e.g., a mammal such as a human patient
  • the antiserum contains antibodies that specifically target the Kexin protein or a Kex peptide thereof to neutralize the activity of Kex proteinase.
  • the isolated antiserum allows the recipient subject to achieve and passively acquire protective immunity against multiple fungal pathogens.
  • a method of the invention involves treating a subject suffering from or susceptible to an infection by Pneumocystis, Aspergillus, Candida, or Cryptococcus, or disease or symptom thereof caused by one or more of these fungal pathogens (e.g., pulmonary disease or COPD).
  • the method includes the step of administering to the subject (e.g., a mammal or human patient) a therapeutically effective amount of an isolated antiserum generated against a Kex peptide of one type of fungal organism that is sufficient to treat an infection, disease, disorder, or symptom thereof, caused by one or more different types of fungal organism under conditions such that the infection, disease, disorder, or symptom thereof, is treated.
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • the present invention also provides methods of treating or preventing infection by more than one type of fungal pathogen, and/or diseases or disorders or symptoms thereof, which comprise administering a therapeutically effective amount of an isolated antiserum as described herein (e.g., comprising one or more antibodies or an antigen binding fragment thereof), to a subject (e.g., a mammal such as a human).
  • a therapeutically effective amount of an isolated antiserum as described herein e.g., comprising one or more antibodies or an antigen binding fragment thereof
  • the method prevents infection by more than one fungal pathogen selected from Pneumocystis,
  • the method includes the step of administering to a recipient mammal a therapeutically effective amount of an isolated antiserum sufficient to treat the infection, disease, disorder, or symptom thereof, under conditions such that the infection, disease, disorder, or symptom thereof is treated.
  • the method includes the step of administering to a recipient mammal a prophylactic or preventive amount of an antiserum sufficient to prevent the infection, disease, disorder, or symptom thereof, under conditions such that the infection, disease, disorder, or symptom thereof is prevented.
  • the isolated antiserum is in a
  • the recipient mammal is a human patient in need of treatment.
  • Treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for infection by, more than one fungal organism, in particular, more than one of Pneumocystis, Aspergillus, Candida, or Cryptococcus, and in particular, Pneumocystis hominis or jirovecii, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans, or a disease, pathogenic condition, or symptom thereof.
  • more than one fungal organism in particular, more than one of Pneumocystis, Aspergillus, Candida, or Cryptococcus, and in particular, Pneumocystis hominis or jirovecii, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans, or a disease, pathogenic condition, or symptom thereof.
  • Determination of those subjects who are "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme test or assay, or protein marker (such as levels of anti-Kex antibodies, e.g., in serum), family history, and the like).
  • the methods herein also include administering to the recipient subject (including a subject identified as in need of such treatment or as being at risk of infection) an effective amount of an anti-fungal pathogen antiserum isolated from a donor subject as described herein.
  • Identifying a subject in need of such treatment can involve the judgment of the recipient subject or a health care or medical professional and can be subjective (e.g., opinion) or objective (e.g., measurable by a test or diagnostic method).
  • the isolated antiserum is provided in a pharmaceutically acceptable
  • the invention features methods of treating or preventing a fungal infection or fungal pathogen-associated disease or condition (e.g., pulmonary infection, pulmonary disease or disorder, pneumonia, COPD, asthma including severe asthma, and the like) in a subject, the methods comprising administering to a subject in need thereof an effective amount of an isolated antiserum obtained from an individual who has produced an antibody immune response against a fungal Kexin protein or immunogenic peptide thereof as described herein, such that the subject is therapeutically and/or prophylactically treated against infection or disease associated with a different fungal pathogen.
  • a fungal infection or fungal pathogen-associated disease or condition e.g., pulmonary infection, pulmonary disease or disorder, pneumonia, COPD, asthma including severe asthma, and the like
  • the invention provides methods of treating or preventing fungal infection and/or disease in a patient who is receiving or who has received immune suppressive drugs or medication and who, as a result of drug-induced immune system suppression, is susceptible to or may become susceptible to (or at risk of) infection by a pathogenic fungus, such as one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens, either in or outside of a nosocomial environment.
  • a pathogenic fungus such as one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens, either in or outside of a nosocomial environment.
  • such a patient may be preparing to undergo a transplant (a pre-transplant patient) or may have received a transplant (a post-transplant patient) and is administered one or more immunosuppressive drugs or medications (anti-rejection medications) and/or is otherwise treated with drugs to reduce the likelihood of rejection of the transplanted organ or tissue, thereby making the patient more vulnerable, susceptible to, or at risk of infection and/or disease caused by one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • immunosuppressive drugs or medications anti-rejection medications
  • Patients having other types of diseases and conditions such as rheumatoid arthritis or psoriasis, and the like, may also be administered medications having an immune suppressive effect to treat or manage their conditions and thus suffer from, or be at risk of, infection by one or more fungal pathogens.
  • Non-limiting classes of immune suppressive drugs and medications include, for example, corticosteroids, such as prednisone (e.g., DELATSONE, ORASONE); budesonide (ENTOCORT EC), or prednisolone (MLLIPRED) calcineurin inhibitors, such as cyclosporine (NEORAL, SANDIMMUNE, SANGCYA); or tacrolimus (ASTAGRAF XL, ENVARSUS XR, PROGRAF); mTOR inhibitors, such as sirolimus (RAPAMUNE), everolimus (AFINITOR, ZORTRESS); Inosine Monophosphate Dehydrogenase (IMDH) inhibitors, such as azathioprine (AZASAN, IMURAN), leflunomide (ARAVA), mycophenolate (CELLCEPT, MYFORTIC); Biologies and monoclonal antibodies or monoclonal antibody-based antibodies or antigen binding fragments thereof, such as abatacept (ORENCIA);
  • certolizumab (CIMZIA); etanercept (ENBREL); golimumab (SIMPONI); infliximab (REMICADE); ixekizumab (TALTZ); natalizumab (TYSABRI); rituximab (RITIXAN); secukinumab (COSENTYX); tocilizumab (ACTEMRA); ustekinumab (STELARA); and vedolizumab (ENTYVIO).
  • the patient is to receive or has received a transplant of an organ selected from kidney, liver, heart, bone marrow, pancreas, lung, gall bladder, bladder, etc.
  • Antibodies directed against the Kex peptides of at least one, or at least two of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens can be administered to the patient who is receiving transplant anti-rejection medication, or other immune suppressive medication, in an effective amount to heighten the immune response against infection by one or more, two or more, three or more, or all four of these fungal pathogens in the immune suppressed patient.
  • the patient receiving immune suppressing drugs can be evaluated and monitored during treatment with immune suppressive drugs for the presence of antibodies (and antibody titers) against one or more of the fungal pathogens by employing the methods and kits as described herein.
  • an isolated antiserum of the invention may be administered in combination with one or more of any other treatment or therapy, e.g., anti-fungal therapies.
  • an isolated antiserum or immune plasma containing mti-Pneumocystis Kex peptide (or mti-Aspergillus, Candida, or Cryptococcus Kex peptide) antibody or an antigen binding fragment thereof as described herein may be administered in combination with other antibodies or antibody cocktails with anti-fungal activity (including, for example, immune plasma), or in combination with one or more drugs, for examples, one or more drugs having anti-fungal activity (e.g., trimethoprim- sulfamethoxazole, azithromycin-sulfamethoxazole, clarithromycin-sulfamethoxazole, atovaquone, sulfadoxine-pyrimethamine, erythromycin-sulfisoxazole, PS-15, and dapsone- trimethoprim, as well as intravenous pentamidine and clindamycin-primaquine), to provide protective immunity in the recipient not only against Pneum
  • an isolated antiserum or immune plasma containing mti-Aspergillus Kexin polypeptide or peptide antibody as described herein may be administered in combination with other antibodies or antibody cocktails with anti-fungal activity (including, for example, immune plasma), or in combination with a drug with anti-fungal activity as described above to provide protective immunity not only against Aspergillus, but also against Pneumocystis, Candida, or
  • an isolated antiserum or immune plasma containing anti-Candida Kexin polypeptide or peptide antibody as described herein may be administered in combination with other antibodies or antibody cocktails with anti-fungal activity (including, for example, immune plasma), or in combination with a drug with anti-fungal activity as described above to provide protective immunity not only against Candida, but also against Pneumocystis, Aspergillus, or Cryptococcus fungal organisms in the subject.
  • an isolated antiserum or immune plasma containing anti- Cryptococcus Kexin polypeptide or peptide antibody as described herein may be administered in combination with other antibodies or antibody cocktails with anti-fungal activity (including, for example, immune plasma), or in combination with a drug with anti-fungal activity as described above to provide protective immunity not only against Candida, but also against Pneumocystis, Aspergillus, or Cryptococcus fungal organisms in the subject.
  • the isolated antiserum is provided in a pharmaceutically acceptable composition.
  • the antiserum isolated from a donor subject immunized, inoculated, or exposed to a Pneumocystis , Aspergillus, Candida, or Cryptococcus fungal pathogen, e.g., a Kex peptide expressed by such fungal pathogens allows the recipient subject to acquire immune protection, including memory immune protection, against infection or disease caused by more than one of these fungal pathogens.
  • Methods for administering both single and combination therapies are known to those skilled in the art and are described, for example, in
  • the antiserum provides a therapeutic, antibody-containing composition that treats infection or disease caused by one or more fungal pathogens as described herein.
  • the antiserum provides prophylactic, antibody - containing composition that prevents and protects against infection or disease caused by one or more fungal pathogens as described herein.
  • the isolated antiserum is in a pharmaceutically acceptable composition.
  • methods are provided that allow a patient to be treated with the appropriate or a more directed fungal therapy by stratifying patients based on whether they possess or do not possess antibodies specific for a particular fungal pathogen, thereby deterring infection, or based on whether they have or do not have antifungal Kex peptide antibodies that are specific for a given type of pathogenic fungus.
  • a method for detecting antibodies against a fungal- derived Kex protein or peptide in a sample obtained from a subject, in which the method comprises: (a) contacting a biological sample obtained from the subject with a Kex peptide derived from one or more, two or more, three or more, or four different fungal organisms selected from Pneumocystis, Aspergillus, Candida, or Cryptococcus; and (b) detecting the binding of the fungal-derived Kex peptide to antibodies in the sample which specifically bind to the Kex peptide, said detection of binding being indicative of the presence of antibodies against the Kex peptide of the fungal organisms in the subject's sample.
  • the Kex peptide is attached to a solid support or substrate.
  • the binding is detected by performing an immunoassay, e.g., an enzyme linked immunosorbent assay.
  • the Kex peptide of one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms is attached to the solid support or substrate.
  • the Kex peptides of two or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms are attached to the solid support or substrate.
  • the Kex peptides of three or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms are attached to the solid support or substrate. In an embodiment, the Kex peptides of all four of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms are attached to the solid support or substrate.
  • a method of monitoring or detecting antibodies to fungal organisms associated with infection in a subject who has undergone a transplant or who is to undergo a transplant procedure to determine, for example, whether the subject is protected or will be protected from infection by one or more fungal pathogens selected from
  • the method comprises: (a) measuring at a first time point the level of antibodies that bind to a Kex protein or peptide derived from at least one, at least two, at least three, or four different fungal organisms selected from Pneumocystis, Aspergillus, Candida, or Cryptococcus, in a sample obtained from the subject prior to undergoing transplant surgery; (b) measuring the levels of antibodies that bind to a Kex protein or peptide derived from at least one, at least two, at least three, or four different fungal organisms selected from Pneumocystis, Aspergillus, Candida, or Cryptococcus in a sample obtained from the subject at one or more time points after the subject has undergone transplant surgery; and (c) detecting that the subject sample contains a level of antibodies that specifically bind to the Kex peptide of one or more of the
  • antibodies detected in the subject's sample that bind to the Kex peptide of Pneumocystis may serve to protect the subject from infection not only by Pneumocystis, but also from infection by one or more of the other fungal organisms, Aspergillus, Candida, or Cryptococcus, according to the present invention.
  • the detection of a high level of antibodies that bind Aspergillus-derived Kex peptide in the subject's sample may serve to protect the subject from infection not only by Aspergillus, but also from infection by one or more of the other fungal organisms, Pneumocystis, Candida, or Cryptococcus, according to the present invention.
  • the detection of a high level of antibodies that bind Candida-dehved Kex peptide in the subject's sample may serve to protect the subject from infection not only by Candida, but also from infection by one or more of the other fungal organisms, Pneumocystis, Aspergillus, or Cryptococcus, according to the present invention.
  • the detection of a high level of antibodies that bind Cryptococcus-derived Kex peptide in the subject's sample may serve to protect the subject from infection not only by Cryptococcus, but also from infection by one or more of the other fungal organisms, Pneumocystis, Aspergillus, or Candida, according to the present invention.
  • Embodiments of the invention also provide methods for detecting in a subject's biological sample, e.g., blood, serum, plasma, lymph, bronchoalveolar lavage fluid, the presence of antibodies that bind Kex peptide derived from one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms, wherein the levels of antibodies against one or more of the fungal organisms in the biological sample are determined simultaneously.
  • a subject's biological sample e.g., blood, serum, plasma, lymph, bronchoalveolar lavage fluid
  • the method comprises: (a) contacting a biological sample obtained from the subject with a plurality of Kex peptides that selectively bind to a plurality of antibodies in the subject's sample for a period of time sufficient to form bound Kex pepti de-antibody complexes; (b) detecting binding of the Kex peptides to the plurality of antibodies in the subject's sample, thereby determining the levels of antibodies to the Kex peptides in the sample; and (c) comparing the levels of the plurality of antibodies in the sample with predetermined threshold values, wherein levels of antibodies that bind to at least one of the plurality of Kex peptides above or below the predetermined threshold values indicates, for example, that the subject has an antibody titer and has generated an immune response against the Kex peptide derived from one or more of the fungal organisms.
  • the subject having a measured antibody response to the Kex peptide is protected from infection and disease associated with one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms.
  • methods for assessing antibodies that bind to the Kex peptide of one or more of the fungal organisms Pneumocystis, Aspergillus, Candida, or Cryptococcus in a subject comprise: (a) contacting a biological sample obtained from the subject with a composition comprising one or more (a plurality of) Kex peptides derived from Pneumocystis, Aspergillus, Candida, or Cryptococcus for a period of time sufficient to form antibody-Kex peptide complexes; (b) detecting binding of the plurality of the Kex peptides to antibodies in the sample, thereby detecting the level or titer of anti- Kex peptide antibodies in the sample; and (c) comparing the level or titer of the anti-Kex peptide antibodies in the biological sample with predetermined threshold values or control values, wherein levels of at least one of the anti-Kex peptide antibodies above or below the predetermined threshold values indicates that the
  • a composition which comprises a solid substrate and a plurality of Kex peptides derived from one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus immobilized on the substrate.
  • a Kex peptide from each of Pneumocystis, Aspergillus, Candida, or Cryptococcus is immobilized at a different, indexable, location on the substrate.
  • a mixture of Kex peptides from two or more, three or more, or all four of Pneumocystis, Aspergillus, Candida, or Cryptococcus is immobilized at a different, indexible, location on the substrate.
  • the binding of anti-Kex peptide antibodies from a sample obtained from a subject can be measured or detected by measuring or detecting complexes of the anti-Kex peptide antibodies bound to the Kex peptides localized on the substrate.
  • the composition is contained in a kit for performing an immunoassay to detect and/or measure the antibody - peptide complexes, as well as determine or measure the level or amount of antibody present in the subject's sample.
  • antisera comprising antibodies that specifically bind a Kexin peptide of a fungal organism, such as Pneumocystis, and that cross-react with a Kexin peptide of one or more different fungal organisms, such as one or more of Aspergillus, Candida, or Cryptococcus, to provide immune protection against infection and disease caused by two or more of these fungal pathogens are useful in therapeutic methods.
  • isolated antiserum containing antibodies that target and/or inhibit or neutralize the activity of the Kexin protein in two or more of these fungal pathogens is particularly useful in the methods of the invention.
  • the invention provides methods of using isolated antiserum (or immune plasma) comprising mti-Pneumocystis, Aspergillus, Candida, or Cryptococcus Kexin polypeptide antibodies or antigen binding fragments thereof, in particular, anti-Kex peptide antibodies or antigen binding fragments thereof, for the treatment or prevention of infection by these fungal pathogens and/or their associated diseases and conditions, such as pulmonary diseases and disorders of various types, pneumonia, COPD, asthma, including severe asthma, etc.
  • the antisera and/or the specific antibodies contained therein may be obtained or isolated from individuals who have recovered from fungal infection caused by one or more of the Aspergillus, Pneumocystis, Candida, or Cryptococcus fungal pathogens or from those who have survived infection caused by one or more of these organisms.
  • the antiserum is obtained or isolated from blood, serum, or plasma of such individuals.
  • antibody means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen-binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term “antibody” means not only intact
  • immunoglobulin molecules but also the well-known active fragments F(ab')2, and Fab.
  • the antibodies of the invention comprise whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.
  • Unconventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al, Protein Eng. 8(10): 1057-1062, (1995)), single domain antibodies, single chain antibodies, and antibodies having multiple valencies (e.g. , diabodies, tribodies, tetrabodies, and pentabodies).
  • Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies have the affinity and specificity of conventional antibodies although they are only half of the size of a single chain Fv fragment. The consequence of this unique structure, combined with their extreme stability and a high degree of homology with human antibody frameworks, is that nanobodies can bind therapeutic targets not accessible to conventional antibodies.
  • Recombinant antibody fragments with multiple valencies provide high binding avidity and unique targeting specificity to cancer cells.
  • These multimeric scFvs e.g. , diabodies, tetrabodies
  • Power et al (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy, Methods Mol Biol, 207, 335-50, (2003); and Wu et al, Anti- carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging, Tumor Targeting, 4, 47-58, (1999)).
  • CEA Anti- carcinoembryonic antigen
  • Bispecific antibodies produced using leucine zippers are described by Kostelny et al. (J. Immunol. 148(5): 1547-1553, (1992)). Diabody technology is described by Hollinger et al. (Proc. Natl. Acad. Sci. USA 90:6444-6448, (1993)). Another strategy for making bispecific antibody fragments using single-chain Fv (sFv) diners is described by Gruber et al. (J. Immunol. 152:5368, (1994)). Trispecific antibodies are described by Tutt et al. (J.
  • Single chain Fv polypeptide antibodies include a covalently linked VH: :VL heterodimer which can be expressed from a nucleic acid including VH- and VL-encoding sequences either joined directly or joined by a peptide-encoding linker as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, (1988)). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754.
  • an antiserum contains anti-Kex peptide antibodies or antigen binding fragments thereof derived from the fungal organisms described herein that are monoclonal or polyclonal.
  • the antiserum or immune plasma containing antibodies that bind Kexin polypeptides and peptides is obtained from an immune survivor of infection by one or more of the fungal pathogens described herein.
  • the antiserum is an antiserum isolated from a survivor of infection by one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • the invention also encompasses obtaining or isolating the antibodies from immune serum
  • hybrid or chimeric antibodies In such hybrid or chimeric antibodies one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody.
  • hybrids or chimeric antibodies may also be formed using humanized heavy and light chains. Methods for isolating antibodies and producing hybrid or chimeric antibodies are known and practiced by those having skill in the art.
  • intact antibodies are said to contain "Fc” and "Fab” regions.
  • the Fc regions are involved in complement activation and are not involved in antigen binding.
  • An antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an "F(ab')2" fragment, retains both of the antigen binding sites of the intact antibody.
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an "Fab"' fragment, retains one of the antigen binding sites of the intact antibody.
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, denoted "Fd.”
  • the Fd fragments are the major determinants of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to immunogenic epitopes.
  • Antibodies can be produced or generated by any of the methods known in the art utilizing soluble polypeptides, or immunogenic fragments thereof, (e.g., a Kex peptide) as an immunogen.
  • One method of obtaining antibodies is to immunize suitable host animals or subjects with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production.
  • the immunogen will facilitate presentation of the immunogen (or immunogenic fragments of the immunogen) on the cell surface. Immunization of a suitable host can be carried out in several ways.
  • nucleic acid sequences encoding immunogenic Kexin peptides can be provided to the host in a delivery vehicle (or a molecular expression construct) that is taken up by immune cells of the host.
  • the cells will, in turn, process and appropriately express the Kex peptide in a manner that generates an immunogenic response in the host.
  • Kex peptides of Pneumocystis, Aspergillus, Candida, or Cryptococcus may be expressed by the delivery vehicle or expression construct, e.g., as shown in FIG. 4.
  • nucleic acid sequences encoding Kex peptides of Pneumocystis, Aspergillus, Candida, or Cryptococcus may be expressed in cells in vitro, and the expressed, recombinant Kex peptide products may be isolated and used as immunogens to raise anti-Kex peptide antibodies and to generate an anti- Kex antiserum in a suitable immunized host.
  • antibodies against the Kex peptides of any, or all, of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens may, if desired, be derived from an antibody phage display library.
  • a bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human immunoglobulin (antibody) genes, to display human antibody proteins.
  • Phage display is the process by which the phage is made to 'display' the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.
  • Antibodies made by any method known in the art can then be purified from an immunized host.
  • Antibody purification methods include, without limitation, salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column, preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC) and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, or anti-immunoglobulin.
  • salt precipitation for example, with ammonium sulfate
  • ion exchange chromatography for example, on a cationic or anionic exchange column, preferably run at neutral pH and eluted with step gradients of increasing ionic strength
  • gel filtration chromatography including gel filtration HPLC
  • affinity resins such as protein A, protein G, hydroxyapatite, or anti-immunoglobulin.
  • antibodies can be conveniently produced from hybridoma cells engineered to express the antibody.
  • Methods of making hybridomas are well known in the art.
  • the hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source.
  • Polynucleotides encoding the antibody of interest can, in turn, be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these nucleic acid sequences.
  • the method of raising ascites fluid generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse.
  • the mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane).
  • a suitable composition e.g., Pristane
  • Ascites fluid containing antibodies, typically in high concentration, can be obtained from the peritoneal fluid of the animal that harbors the injected hybridoma cells.
  • Monoclonal antibodies can also be “humanized” by methods known in the art.
  • “Humanized” antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like immunoglobulins derived from a human source. Techniques to humanize antibodies are particularly useful when antibodies are generated in a non-human animal (e.g. , mice, rats). Nonlimiting examples of methods for humanizing a murine antibody are provided in U.S. Patent Nos 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205.
  • one or more antibodies or antigen binding fragments thereof generated against the Kex peptide derived from the different fungal pathogens can be used in a pharmaceutical composition alone or in combination to provide immune protection against disease or infection caused by one or more of these fungal pathogens in a subject in need thereof.
  • Such antibodies may be isolated or purified from an antiserum as described herein, or they may be generated, e.g., by recombinant molecular biology techniques, purified and formulated for pharmaceutical use in a subject in need.
  • Such a formulation of antibodies may have immune protective properties similar to those afforded by an isolated antiserum comprising anti-fungal Kex peptide antibodies as described herein.
  • a vaccine is a biological preparation that provides active, acquired immunity in a subject to a particular disease.
  • a vaccine typically contains an agent that resembles a disease-causing pathogenic agent, e.g., a microorganism, a fungus, etc., and is often made from a weakened or killed form of the agent, or a toxin or surface protein or peptide of the agent.
  • the agent is expressed and recognized as foreign (or "non-self) to the subject and stimulates the subject's immune system to mount an immune response (a B cell (antibody) and/or a T cell (cellular) immune response) and to destroy the agent.
  • Vaccines can be prophylactic (e.g., to prevent or ameliorate the effects of a future infection by a pathogen), or therapeutic (e.g., to treat disease or infections caused by or associated with pathogens or disease-causing agents upon or after a subject has been infected with or encountered a pathogen).
  • a protein or peptide part of a pathogen may constitute a vaccine.
  • a peptide vaccine is any peptide which serves to immunize an organism (elicit an immune response or a protective immune response, such as an antibody (B cell) response and/or an immune cell (T cell) response in the immunized organism) against a pathogen.
  • the peptide antigen may be a Kex peptide derived from one, two, three, or all of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens as described herein.
  • a vaccine comprising a Kex peptide antigen derived from a fungal organism, such as a, Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organism described herein, may be used to provide immune protection against each of the other fungal organisms following administration to a recipient subject in need.
  • the peptide sequences that trigger a protective immune response are identified, and synthetic (or recombinantly-produced) versions of the peptides are employed as the vaccine substance. Because they are non-naturally occurring and synthetic, peptide vaccines pose little to no risk of mutation or reversion, and little or no risk of contamination by pathogenic or toxic substances. Moreover, chemical manipulation or modification of the peptide structure may result in increased stability and decreased unwanted side effects or adverse effects that may be associated with a native protein or peptide sequence.
  • Synthetically or recombinantly produced peptide antigens can be readily prepared in large amounts as components of vaccines. Such substances may also expose parts of a protein antigen that are not recognized by the immune system during a natural infection, possibly as a result of masking or post-translational modifications of proteins. Sequencing new strains and serotypes of microorganisms, fungal pathogens and other pathogenic organisms allows for rapid modification of peptide antigens to generate strain-specific immune responses, particularly against an antigenic epitope that is recognized and targeted by antibodies and cells of the host's or recipient's immune system. In some cases, modelling of three-dimensional epitopic or antigenic sites of a pathogen may be employed to generate synthetically the correct epitopic or antigenic site(s) on peptide antigens.
  • the invention embraces a vaccine (or an immunogenic composition) comprising a synthetically (recombinantly) produced peptide, i.e., a Kexin peptide, that is nonidentical, but immunologically targetable, among several different types of fungal pathogens (e.g., the Pneumocystis , Aspergillus, Candida and Cryptococcus fungal pathogens) and is useful for treating or preventing infection or disease caused by or associated with the multiple fungal pathogens after administration (immunization) to a subject.
  • Kexin peptide sequences for use in generating an immune response or antibody response are shown, for example, in FIGS. 1 and 4.
  • a peptide vaccine or immunogenic composition from one of Pneumocystis, Aspergillus, Candida, or
  • Cryptococcus and in particular, Pneumocystis hominis, Aspergillus fumigatus, Candida albicans or Cryptococcus neoformans fungal organisms, when used to immunize an individual, elicits an immune response in the form of the production of antiserum (or immune plasma) containing cross-reactive antibodies which protect against all of the etiologically distinct fungal pathogens Pneumocystis, Aspergillus, Candida and Cryptococcus, and in particular, Pneumocystis hominis, Aspergillus fumigatus, Candida albicans and Cryptococcus neoformans.
  • an antiserum or immune plasma generated by a vaccine or immunogenic composition comprising a Kex peptide of a Pneumocystis, Aspergillus,
  • Candida or Cryptococcus (or from a Pneumocystis hominis, Aspergillus fumigatus, Candida albicans, or Cryptococcus neoformans fungal organism as described herein) may be used as a sole therapeutic or protective agent needed to treat or prevent infection or disease caused by or associated with infection not only by the fungal organism whose Kex peptide was used as immunogen, but also by more than one different fungal pathogen, namely, the Pneumocystis, Aspergillus, Candida and/or Cryptococcus fungal pathogens, and in particular, infection or disease caused by or associated with Pneumocystis hominis, Aspergillus fumigatus, Candida albicans and/or Cryptococcus neoformans.
  • the antiserum generated by such as peptide vaccine is isolated.
  • the isolated antiserum is used in a pharmaceutical composition.
  • the present invention features methods for treating or preventing infection and disease associated with infection of a subject by one or more fungal pathogens.
  • the methods include administering to a subject in need thereof an immunologically effective amount of an isolated antiserum containing antibodies generated against a Kexin peptide derived from a Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogen, which antiserum cross-treats and/or protects the subject from infection by and/or disease associated with not only the fungal organism from which the Kex peptide immunogen was derived, but also from infection by and/or disease associated with one or more different fungal pathogens selected from Pneumocystis, Aspergillus, Candida, or Cryptococcus.
  • the isolated antiserum is used in a pharmaceutical composition.
  • the carrier or excipient for an immunogenic composition or vaccine as described herein is a pharmaceutically acceptable carrier or excipient, such as sterile water, aqueous saline solution, aqueous buffered saline solutions, aqueous dextrose solutions, aqueous glycerol solutions, ethanol, or combinations thereof.
  • a carrier or excipient is selected to minimize allergic and other undesirable effects, and to suit the particular route of administration, e.g. , subcutaneous, intramuscular, intranasal, and the like.
  • Such methods also include administering an adjuvant, such as an oil-in-water emulsion, a saponin, a cholesterol, a phospholipid, a CpG, a polysaccharide, variants thereof, and a combination thereof, with the composition of the invention.
  • an adjuvant such as an oil-in-water emulsion, a saponin, a cholesterol, a phospholipid, a CpG, a polysaccharide, variants thereof, and a combination thereof
  • a formulation for prophylactic administration also contains one or more adjuvants for enhancing the immune response to an antigen or immunogen, such as a Kex peptide antigen or immunogen.
  • Suitable adjuvants include, without limitation, complete Freund's adjuvant, incomplete Freund's adjuvant, saponin, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil or hydrocarbon emulsions, bacille Calmette-Guerin (BCG), Corynebacterium parvum, and the synthetic adjuvants QS-21 and MF59 (Novartis).
  • the isolated antiserum is used in a pharmaceutical composition.
  • an antiserum such as an isolated antiserum, or anti- Pneumocystis, Aspergillus, Candida, and/or Cryptococcus Kexin protein or peptide monoclonal or polyclonal antibodies isolated and purified from a non-immunized, but naturally immune protected, subject (donor subject), as a therapeutic for the treatment or prevention of an infection by a fungal pathogen as described herein or a fungal pathogen- associated disease or condition (e.g., pulmonary infection or disease, poor pulmonary function, COPD, pneumonia, etc.) may be by any suitable means that results in a fungal pathogen as described herein or a fungal pathogen- associated disease or condition (e.g., pulmonary infection or disease, poor pulmonary function, COPD, pneumonia, etc.) may be by any suitable means that results in a fungal pathogen as described herein or a fungal pathogen- associated disease or condition (e.g., pulmonary infection or disease, poor pulmonary function, COPD, pneumonia, etc
  • the therapeutic may be administered systemically, for example, formulated in a pharmaceutically-acceptable composition or buffer such as physiological saline.
  • Preferable routes of administration include, for example, subcutaneous, intravenous, intraperitoneally, intramuscular, intrathecal, or intradermal injections that provide continuous, sustained levels of the therapeutic in the subject.
  • the amount of the therapeutic to be administered varies depending upon the manner of administration, the age and body weight of the subject, and with the clinical symptoms of the fungal infection or associated disease.
  • amounts will be in the range of those used for other agents used in the treatment of pulmonary disease or dysfunction, although in certain instances lower amounts may be suitable because of the increased range of protection and treatment afforded by the therapeutic.
  • a composition is administered at a dosage that ameliorates, decreases, diminishes, abates, alleviates, or eliminations the effects of the fungal pathogen infection or disease (e.g., pulmonary infection and disease and the symptoms thereof) as determined by a method known to one skilled in the art.
  • the isolated antiserum is administered or provided to a recipient subject at or near a site of the infection or colonization by the pathogenic fungal organism or organisms.
  • a therapeutic or prophylactic treatment agent may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1 -95% by weight of the total weight of the composition.
  • the composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal) administration route.
  • parenteral e.g., subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal
  • the pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J.
  • compositions may in some cases be formulated to release the active agent substantially immediately upon administration or at any predetermined time or time period after administration.
  • controlled release formulations include (i) formulations that create a substantially constant concentration of a therapeutic agent or drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of a therapeutic agent or drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (sawtooth kinetic partem); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in contact with an organ, such as the heart; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target a controlled release composition adjacent to or in contact with
  • controlled release formulations obviate the need for frequent dosing during the day to sustain a therapeutic level in plasma, serum, or blood.
  • an isolated antiserum may be formulated with one or more additional components for administration to a subject. Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the therapeutic agent or drug in question.
  • controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings.
  • the therapeutic agent or drug may be formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic agent or drug in a controlled manner. Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.
  • a pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • injection, infusion or implantation subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, or the like
  • suitable delivery devices or implants containing conventional, non-toxic pharmaceutically acceptable carriers and adjuvants.
  • Formulations can be found in Remington: The Science and Practice of Pharmacy, noted supra.
  • compositions for parenteral use may be provided in unit dosage forms (e.g., in single- dose ampules), or in vials containing several doses and in which a suitable preservative may be added (see below).
  • the composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use.
  • the composition may include suitable parenterally acceptable carriers and/or excipients.
  • an active therapeutic agent(s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release.
  • composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents.
  • a pharmaceutical composition comprising an active therapeutic (e.g., an isolated anti-fungal antiserum described herein) is formulated for intravenous delivery, e.g., intravenous, injection, or intrathecal delivery.
  • an active therapeutic e.g., an isolated anti-fungal antiserum described herein
  • the antiserum is an isolated antiserum.
  • the suitable therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle, excipient, or solvent.
  • acceptable vehicles and solvents that may be employed are, for example, water; water adjusted to a suitable pH by the addition of an appropriate amount of hydrochloric acid, sodium hydroxide or a suitable buffer; 1,3-butanediol; Ringer's solution; and isotonic sodium chloride solution and dextrose solution.
  • the aqueous formulation may also contain one or more preservatives (e.g., methyl, ethyl or n-propyl p-hydroxybenzoate).
  • a dissolution enhancing or solubilizing agent can be added, or the solvent may include 10-60% w/w of propylene glycol or the like.
  • kits and compositions are provided that advantageously allow for the detection and/or quantification of the presence of antibodies directed against the Kex protein or peptide of one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens, or the levels of such one or more antibodies that may be present, in a subject's sample (e.g., blood or serum).
  • a subject's sample e.g., blood or serum
  • the subject is a human patient.
  • the patient has undergone a transplant, e.g., an organ or tissue transplant, or is to undergo a transplant, and thus may be at higher risk for infection by one or more fungal pathogens.
  • the transplant patient or the patient to undergo a transplant, is immunosuppressed and/or is otherwise treated with drugs to reduce the likelihood of rejection of the transplanted organ or tissue, thereby making the patient more vulnerable or susceptible to infection and/or disease caused by one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • the patient has received, or is to receive, a transplant of an organ selected from kidney, liver, heart, bone marrow, pancreas, lung, etc.
  • kits as described herein fulfill a long-felt need in the art for detecting or qualifying whether any patient, but particularly a transplant patient, has adequate levels (titer) of anti-fungal pathogen antibodies to ensure that the patient does not become infected with one or more fungal pathogens as described herein, for example, during a hospital stay, or during or following a medical procedure or treatment (e.g., surgery or transplant), performed either on in-patient or an out-patient basis.
  • a medical procedure or treatment e.g., surgery or transplant
  • a patient who is to undergo a medical procedure or surgery in particular, an immunosuppressed patient who is to undergo a transplant procedure, or a patient who is to initiate other immunosuppressive therapies, will contract a fungal infection, e.g., infection and/or disease caused by one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens following or during immunosuppressive therapies and treatments.
  • a fungal infection e.g., infection and/or disease caused by one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens following or during immunosuppressive therapies and treatments.
  • kits with which a patient's sample can be tested to determine if the patient has an antibody titer against one or more of these fungal pathogens would greatly enhance the success of the patient's post-surgical or post-transplant recovery and directed treatment.
  • a patient's sample e.g., a blood or serum sample from a transplant patient
  • the patient is determined to have a low, negligible, or no antibody (antiserum) titer against against one or more of the fungal pathogens, in particular, against the Kex peptide of one or more of the fungal pathogens, it could be surmised that the patient would not be naturally protected against a possible or real infection by one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus.
  • kits as described herein would allow the tester and the patient to determine and know if the patient's sample (serum sample) contains antibodies against one or more, two or more, three or more, or four of Kex protein/peptide of the Pneumocystis, Aspergillus,
  • the patient would be identified as potentially vulnerable or susceptible to infection to (a) particular fungal pathogen(s) and could then be administered the appropriate anti-fungal treatment for the specific fungal pathogen(s) against which the patient has no, or negligible, specific antibodies, or a reduced antibody titer.
  • the patient is administered a prophylactic anti-fungal treatment or therapy.
  • the treatment comprises administering to the patient an appropriate drug or medication that is best designed to treat infection or disease associated with infection by a specific fungal pathogen or by two or more fungal pathogens, namely, Pneumocystis, Aspergillus, Candida, or Cryptococcus.
  • the treatment comprises administering to the patient a composition as described herein comprising one, two, three, or four of the described fungal-derived Kex peptides to generate a cross-reactive (cross- protective) antibody immune response in the patient, thereby reducing or eliminating one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus organisms associated with the infection.
  • Antibodies produced against a Kex peptide of one of the fungal organisms can recognize a Kex peptide of the other fungal organisms as described herein, thereby conferring protection (cross-protection) against more than one of the fungal organisms in the patient.
  • the invention provides a kit for detecting, or qualifying the levels of, antibodies directed against the Kex protein or peptide of one, two, three, or four of Pneumocystis, Aspergillus, Candida, or Cryptococcus organisms in a patient sample
  • the kit comprises a substrate having attached thereto a Kex peptide derived from one or more, two or more, three or more, or each of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms for measuring the level of antibodies in the sample which bind or immunologically react with the peptide, onto which a biological sample obtained from a patient is applied; and a labeled detection molecule for detecting and measuring the level of antibodies that bind to the Kex peptides on the substrate.
  • detecting anti-fungal Kex peptide antibodies in the sample or the measuring the level of such antibodies present in the patient's sample is compared to a positive and/or a negative control. In an embodiment, detecting anti-fungal Kex peptide antibodies in the sample or the measuring the level of such antibodies present in the patient's sample is compared to a cutoff value.
  • the substrate has attached thereto Kex peptide derived from each of Pneumocystis, Aspergillus, Candida, and Cryptococcus. In an embodiment, the substrate has attached thereto Kex peptide derived from one of Pneumocystis, Aspergillus, Candida, and Cryptococcus.
  • the substrate has attached thereto Kex peptide derived from Pneumocystis and Aspergillus. In an embodiment, the substrate has attached thereto Kex peptide derived from Pneumocystis and Candida. In an embodiment, the substrate has attached thereto Kex peptide derived from Pneumocystis and Cryptococcus. In an embodiment, the substrate has attached thereto Kex peptide derived from Candida and Aspergillus. In an embodiment, the substrate has attached thereto Kex peptide derived from Cryptococcus and Aspergillus. In an embodiment, the substrate has attached thereto Kex peptide derived from Candida and Cryptococcus.
  • the substrate has attached thereto Kex peptide derived from Pneumocystis, Aspergillus and Candida. In an embodiment, the substrate has attached thereto Kex peptide derived from Pneumocystis, Aspergillus and Candida. In an embodiment, the substrate has attached thereto Kex peptide derived from Pneumocystis, Aspergillus and Cryptococcus. In an embodiment, the substrate has attached thereto Kex peptide derived from Pneumocystis, Cryptococcus and Candida. In an embodiment, the substrate has attached thereto Kex peptide derived horn Aspergillus, Cryptococcus and Candida.
  • the Kex peptides are recombinantly produced.
  • the detection of antibodies in the sample that bind to the fungal-derived Kex peptide is performed using an immunoassay, such as an ELISA.
  • the ELISA detects a complex between a Kex peptide bound to an anti-fungal Kex peptide antibody present in the sample.
  • the Kex peptides are recombinantly produced.
  • the detection of antibodies in the sample that bind to the fungal-derived Kex peptide is performed using an immunosorbent assay, by immunoprecipitation, by immunoblotting, or a combination thereof.
  • kits comprising reagents that allow for assessing, measuring, evaluating or detecting antibodies directed against one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • Such antibodies may be contained in a biological sample obtained from a subject undergoing testing, assessment, or evaluation using the kit.
  • the biological sample may be a blood, serum, plasma, urine, cerebrospinal fluid, sputum, bronchiolar lavage, tears, saliva, or semen sample, or tissue or cell sample obtained from a subject.
  • the reagents of the kit comprise at least one of the Kex peptides of the Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens as described herein, (e.g., FIG. 1A and FIG. 3).
  • the kit is provided as an enzyme linked immunosorbent assay (ELISA) kit comprising the Kex peptides of Pneumocystis, Aspergillus, Candida, or Cryptococcus attached to a solid support or substrate.
  • the peptides thus perform as "capture" reagents that bind to antibodies present in the sample obtained from a subject undergoing testing.
  • the ELISA kit may comprise a solid support, such as a chip, microliter plate comprising many wells (e.g., a 96-well plate), bead, or resin having the peptide capture reagents attached thereon.
  • the kit comprises a Kex peptide derived from each of Pneumocystis, Aspergillus, Candida, or Cryptococcus as described herein attached independently to discrete areas or components of solid substrates or supports, for example, the Kex peptides of each fungal organism are attached to separate and discrete wells of a microtiter plate or are independently attached to beads to produce populations of beads having the Kex peptides from each of Pneumocystis, Aspergillus, Candida, or Cryptococcus attached.
  • the kit comprises a combination or mixture of the Kex peptides derived from Pneumocystis, Aspergillus, Candida, or
  • the Kit comprises a combination of one, two or more, three or more, or four of the Kex peptides derived from Pneumocystis, Aspergillus, Candida, or Cryptococcus attached to a given area of a solid substrate or support, such as a single well of a microtiter plate.
  • a well of a microtiter plate may have attached thereto a Pneumocystis Kex peptide, an Aspergillus Kex peptide, a Candida Kex peptide, or a Cryptococcus Kex peptide.
  • a well of a microtiter plate may have attached thereto a Pneumocystis Kex peptide and an Aspergillus Kex peptide.
  • a well of a microtiter plate may have attached thereto a Pneumocystis Kex peptide and a Candida Kex peptide.
  • a well of a microtiter plate may have attached thereto a Pneumocystis Kex peptide and a Cryptococcus Kex peptide.
  • a well of a microtiter plate may have attached thereto an Aspergillus Kex peptide and a Candida Kex peptide.
  • a well of a microtiter plate may have attached thereto an Aspergillus Kex peptide and a Cryptococcus Kex peptide.
  • a well of a microtiter plate may have attached thereto a Candida Kex peptide and a Cryptococcus Kex peptide.
  • an individual well of a microtiter plate may have attached thereto a Pneumocystis Kex peptide, an Aspergillus Kex peptide and a Candida Kex peptide.
  • an individual well of a microtiter plate may have attached thereto a Pneumocystis Kex peptide, an Aspergillus Kex peptide and a Cryptococcus Kex peptide.
  • an individual well of a microtiter plate may have attached thereto a Pneumocystis Kex peptide, a Candida Kex peptide and a Cryptococcus Kex peptide.
  • an individual well of a microtiter plate may have attached thereto an Aspergillus Kex peptide, a Candida Kex peptide and a Crytococcus Kex peptide.
  • the kit may further comprise a means for detecting the peptides or any antibodies bound thereto, e.g., detectable antibodies, a secondary antibody-signal complex, such as horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody or tetramethyl benzidine (TMB) as a substrate for HRP.
  • a secondary antibody-signal complex such as horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody or tetramethyl benzidine (TMB) as a substrate for HRP.
  • HRP horseradish peroxidase
  • TMB tetramethyl benzidine
  • the kit may be provided as an immunochromatography strip comprising a membrane on which the one, two, three, or four Kex peptides are immobilized, either at discrete loci on the membrane or in combination at one locus of the membrane, and a means for detecting the binding of antibody in a test sample, e.g., detectably labeled peptides, or gold particle bound secondary antibodies, in which the membrane may be a nitrocellulose- based (NC) membrane, a PVDF membrane, or other suitable type of membrane used in the art.
  • a membrane e.g., detectably labeled peptides, or gold particle bound secondary antibodies
  • the membrane may be a nitrocellulose- based (NC) membrane, a PVDF membrane, or other suitable type of membrane used in the art.
  • the kit may comprise a plastic plate or substrate onto which a sample is applied and immobilized detection agents, such as detectably labeled Kex peptides, e.g., gold particle- bound peptides temporally spaced and immobilized on the substrate, e.g., a glass fiber filter or a nitrocellulose membrane, or a labeled detection agent that can detect a complex of antibody bound to Kex peptide in one or more bands on the substrate.
  • a continuous capillary flow of sample e.g., blood or serum
  • sample antibody bound to labeled Kex peptide or sample antibody complexed to Kex peptide reagent may be detected.
  • ELISA assays and immunosorbent assays including ELISA membrane-based
  • immunosorbent assays as well as variations of these assays, are known and practiced by those having skill in the art.
  • Solid or solid phase substrates, or carriers, that can be effectively used in such assays are well known to those of skill in the art and include, for example, 96 well microtiter plates, glass, paper, and microporous membranes constructed, for example, of nitrocellulose, nylon, polyvinylidene difluoride, polyester, cellulose acetate, mixed cellulose esters and
  • Suitable microporous membranes include, for example, those described in U.S. Patent Application Publication No. US 2010/0093557 Al. Methods for the automation of immunoassays are well known in the art and include, for example, those described in U.S. Patent Nos. 5,885,530, 4,981,785, 6,159,750 and 5,358,691.
  • a multiplex assay such as a multiplex ELISA
  • a multiplex ELISA can be used to detect simultaneously different specific antibodies in a test sample.
  • such methods employ an array, wherein multiple binding agents (for example capture antibodies) specific for multiple antibodies are immobilized on a substrate, such as a membrane, with each capture agent being positioned at a specific, pre-determined, location on the substrate.
  • arrays wherein multiple binding agents (for example capture antibodies) specific for multiple antibodies are immobilized on a substrate, such as a membrane, with each capture agent being positioned at a specific, pre-determined, location on the substrate.
  • multiplex arrays can be used in several different formats.
  • flow cytometric multiplex arrays also known as bead-based multiplex arrays, include the Cytometric Bead Array
  • a multiplex ELISA from Quansys Biosciences (Logan, UT) involves coating multiple specific capture reagents at multiple spots (one reagent at one spot) in the same well on a 96-well microtiter plate. Chemiluminescence technology is then used to detect multiple antibodies that bind at the corresponding spots on the plate.
  • a patient can be diagnosed by adding a biological sample
  • the method comprises: (i) collecting blood or serum sample from the subject; (ii) adding subject's sample to the components in the kit, e.g., a holding tube or a substrate; and (iii) detecting the peptide reagents to which the sample antibodies have bound.
  • the subject's sample e.g., blood or serum
  • the Kex peptide reagent(s) is brought into contact with the Kex peptide reagent(s).
  • the antibodies will bind to the Kex peptide reagents, or a subset thereof.
  • blood is not collected from the patient (i.e., it is already collected), and is assayed for the presence of antibodies against the Kex peptide of one or more, two or more, three or more, or four of the Pneumocystis, Aspergillus, Candida, or Cryptococcus organisms using the kit.
  • the sample may comprise a tissue sample or a clinical sample, which can be processed, e.g., homogenized and/or suspended in medium or buffer, prior to assay.
  • any antibody(ies) found to be present in a test sample from a subject may be isolated, or isolated and purified, and further characterized.
  • the kit can also comprise a washing solution or instructions for making a washing solution, in which the combination of the capture reagents and the washing solution allows capture of anti-Kex antibodies on the solid support for subsequent detection by, e.g., secondary antibodies, labeled reagent peptides, or mass spectrometry.
  • a kit can comprise instructions for suitable operational parameters in the form of a label or separate insert (package insert). For example, the instructions may inform a consumer or user about how to collect the sample, how to wash the anti-Kex peptide antibody and Kex peptide reagent complex after binding has occurred, how to interpret the results, etc.
  • the kit can comprise one or more containers with appropriate positive and negative controls or control samples, to be used as standard(s) for detection, calibration, or normalization.
  • kits for the treatment or prevention of an infection or disease caused by or associated with two or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens in another aspect, provides kits for the treatment or prevention of an infection or disease caused by or associated with two or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal pathogens.
  • the kit includes an effective amount of a therapeutic or prophylactic antiserum, which contains anti-Kex peptide antibodies or antigen binding fragments thereof that bind/react with one or more of
  • Pneumocystis Aspergillus, Candida, or Cryptococcus, in unit dosage form.
  • Aspergillus Aspergillus
  • Candida Aspergillus
  • Cryptococcus in unit dosage form.
  • the antiserum is an isolated antiserum.
  • the kit includes a therapeutic or prophylactic composition containing an effective amount of an anti-fungal immunoprotective agent such as antiserum in unit dosage form.
  • the kit comprises a device (e.g. , nebulizer, metered-dose inhaler) for dispersal of the composition or a sterile container which contains a pharmaceutical composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.
  • a pharmaceutical composition of the invention is provided together with instructions for administering the pharmaceutical composition containing isolated antiserum to a subject having or at risk of contracting or developing a fungal infection, particularly infection and disease and the symptoms thereof caused by Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms.
  • the instructions will generally include information about the use of the composition for the treatment or prevention of an infection by one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms.
  • the instructions include at least one of the following: description of the therapeutic/prophylactic agent; dosage schedule and administration for treatment or prevention of infection or symptoms thereof caused by one or more of Pneumocystis, Aspergillus, Candida, or Cryptococcus fungal organisms; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology;
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • Example 1 Identification of fungal vaccine candidates based on Kexl peptide sequence identity
  • Pneumocystis KEX 1 was compared with that of Aspergillus fumigatus Kexin (Accession no.
  • FIGS. 1A, IB and 2 Based on the analysis of a conserved, 99 amino acid region of the Kexin protein (FIGS. 1 A, IB and 3), the corresponding DNA sequences were cloned in an E. coli expression vector and recombinant proteins of the 88-amino acid ⁇ Aspergillus Kex and Candida Kex constructs), and a l l 7-amino acid ⁇ Cryptococcus Kex construct) were produced and purified (FIG. 3).
  • Aspergillus KEX (AF_KexDS88) is a target of humoral immune responses during
  • This example describes a protocol for purifying a recombinantly produced (pET28b vector), (Millipore-Sigma, US), macaque-derived Kexl protein (peptide) that is histidine tagged.
  • LB Lysogeny Broth growth medium with kanamycin (40 ⁇ g/mL), typically in a 1 L volume, pH to 7.5. 10 g NaCl, 5 g Yeast Extract and 10 g Tryptone Peptone are admixed; the volume is brought to 1 L with distilled/deionized H2O.
  • Extraction buffer (sterile filtered), Do not pH, containing 6 M guanidinium, 50 mM Sodium phosphate (Na2HP04, dibasic only) and 300 mM sodium chloride.
  • Wash buffer (sterile filtered), Do not pH, containing 50 mM Sodium phosphate (Na2HP04, Dibasic Only) and 300 mM sodium chloride.
  • PIC His-tag protease inhibitor cocktail
  • Bovine serum albumin (BS A) (100 ⁇ g/mL stock).
  • TEMED N. N,N,N',N'-Tetramethylethylenediamine
  • d. Place the casting block into the holder and set the black plastic plugs into the holder. Turn plugs to depress casting block into the black rubber mat of the holder. Confirm that the bottom of the plates is well sealed by the rubber of the holder in order to avoid leaks.
  • e. Prepare separating/running gel according to the recipe below for a 15%-SDS PAGE gel and add solution to the cavity between aluminum backing plate and glass cover plate. Allow -1.5-2 cm of space at the top of the sandwiched plates for stacking gel. Add ⁇ 1 mL of dLhO to casting block. The gel will begin to polymerize once the APS/ TEMED are added to the solution.
  • fraction concentration is ⁇ 165 ⁇ g/ mL, use 22.5 of sample + 7.5 ⁇ 4X Sample Buffer;
  • Plasmids were transformed into Escherichia coli BL21 (DE3) cells and plated on LB agar supplemented with lOOug/ml ampicillin to select for transformed clones.
  • Kex proteins preparation expression hosts were grown overnight with shaking at 37°C in Luria broth (LB) supplemented with 100 ⁇ g/mL ampicillin and then sub-cultured 1:20 for 2 hours at 37°C, and protein expression was induced by the addition of ImM IPTG, with further incubation for 4 hours at 37°C.
  • LB Luria broth
  • MBP maltose binding protein
  • Rhesus macaque (monkey subject number 1791 1) was intramuscularly immunized with 100 ⁇ g of Pneumocystis KEX1 (purified as described in Example 2 above) and aluminum hydroxide (Imject Alum, Thermo Scientific) mixed in a 1 : 1 ratio and then boosted with 12 weeks later with 50 ⁇ g of KEX1 and alum. Plasma was collected 2 weeks after the boost, and heat-inactivated at 56°C prior to use in immunoblotting. Plasma from a non-immunized monkey (monkey number 8015) was collected and used as a negative control. Reciprocal endpoint titers (RET) from monkey subject number 17911 two weeks post boost were >2xl0 6 .
  • RET Reciprocal endpoint titers
  • reciprocal endpoint titer is the reciprocal of the highest analyte (e.g., antibody or antiserum) dilution that gives a reading above the cutoff value.
  • Control plasma RET was ⁇ 100, as determined by KEX1 -specific ELISA, as described in Kling, H.M. and Norris, K.A., 2016, J. Infect. Dis., 213, 1586-1595.
  • antibody titers in particular, anti-fungal KEX peptide antibodies
  • normal human sera with undetectable absorbance at OD450 i.e., equal to or less than the absorbance measured at OD450 for dilution buffer alone
  • a KEX-ELIS A assay at a dilution of 1 : 100 were used as negative controls.
  • RET Pneumocystis KEXl reciprocal endpoint antibody titers
  • Pneumocystis KEXl antisera cross-reactivity with other fungal KEX recombinant proteins To determine the nature of immunologic cross-reactivity among fungal KEX recombinant proteins, antisera from Pneumocystis KEXl immunized monkeys were tested for reactivity by Western blotting using recombinant KEX proteins following purification and TEV cleavage of the fusion partner. Proteins were resolved by 15% SDS/PAGE and transferred to 0.2 ⁇ nitrocellulose membranes.
  • Membranes were then blocked overnight at 4°C in 5% BSA / 5% non-fat dry milk in PBS-T (0.05% Tween-20) and then incubated with high titer antiserum from monkey number 1791 1 (dilution 1 :500) or low titer antiserum from monkey number 8015 (dilution 1 :500) for 2 hours at room temperature to test for construct cross-reactivity. Blots were then incubated with secondary antibody goat anti-monkey IgG (H+L)-HRP (1: 10,000) in blocking buffer for 1 hour at room temperature and visualized with SuperSignal West Pico Chemiluminescent Substrate (ThermoFisher).
  • MBP maltose binding protein
  • This example describes a protocol for performing an ELISA immunoassay utilizing a recombinantly produced (pET28b vector), Kexl protein (peptide) that is histidine tagged and purified as described above in Example 2.
  • the ELISA is conducted to detect (and quantify) the presence of anti-fungal Kex peptide antibodies in a sample, e.g., blood, plasma, serum, bronchoalveolar lavage, or biological fluid sample.
  • the anti-Kex peptide antibodies to be detected (and quantified) can be directed against, reactive with and/or bind to the Kex peptide of one or more of the Pneumocystis, Aspergillus, Candida, or Cryptococcus Kex peptides.
  • Blocking buffer 5% skim milk in lxPBS
  • F. Secondary Antibody Goat anti-human immunoglobulin-conjugated horseradish peroxidase (1: 10,000 for IgG; Sigma- Aldrich).
  • Negative/normal control plasma with undetectable absorbance at OD450 i.e., equal to or less than dilution buffer alone
  • KEX-ELISA a dilution of 1: 100 is used as negative controls.
  • TMB 3,3',5,5'-Tetramethylbenzidine peroxidase substrate
  • wash plates 2x with wash buffer PBS 0.05% Tween-20 ( ⁇ 200uL wash buffer per well for each wash, flicking and tapping plate between washes).
  • Option 1 Heat-inactivate entire aliquot at 56°C for 30 minutes.
  • Option 2 If heat inactivation of the plasma sample would be detrimental to other potential uses, thaw sample at 4°C or on ice. Remove an aliquot (-100 uL), transfer to a new tube, and heat inactivate (30min, 56°C). Return the remaining sample to the -80°C freezer, noting that it has been thawed lx. c. Centrifuge sample at >10,000g for 1-2 minutes to pellet aggregates prior to use.
  • Dilute plasma 1 100 in blocking buffer. Add 50 of diluted plasma and make serial 2x (or 4x, if needed) dilutions directly in the plate (final volume in each well should be 50 ⁇ ) for generation of endpoint titers (see, FIGS. 7A and 7B). Perform assay in duplicate; set up enough plates for all isotypes of interest, e.g., if there are 10 samples and endpoint titers are to be generated for both IgG and IgM-KEXl antibodies, this would require setting up 4 plates (duplicate plates for both IgG and IgM). Include a negative/normal control on each plate. Cover plates with plate sealers and incubate O/N at 4°C.
  • KEX1 OD450 at a 1 : 100 dilution should be not more than 0.1; however, the lower the OD of the normal/negative control plasma, the better the control is.
  • the same normal/negative control should be used on all plates following the selection of an appropriate normal/negative control.
  • the endpoint titer is defined by the dilution at which the test sample gives the same OD reading as that of the negative control (i.e., where the lines meet). Generally, the reciprocal endpoint titer is reported; thus, if the dilution is 1 : 1600, the endpoint titer is reported as 1600.
  • RET reciprocal endpoint titers
  • average the titers e.g., when doubling-dilutions are made, and a sample from plate 1 has a RET of 1600 and the RET from plate 2 is 3200, then the average titer is 2400). If endpoint titers on duplicate plates do not fall within one dilution of each other, repeat the ELISA on one additional plate and average the 2 values which are closest.
  • the below table presents Optical Density 450 nm (OD450) readings for each dilution (shown as its reciprocal) of a negative/normal control and three representative test samples (951, 962 and 966 in the table), e.g., serum samples.
  • the original OD values for the control sample are plotted on the top row, followed by control values plus 0.025 for standardization of the plate (neg. Ctrl. +
  • Example 7 ⁇ -Aspergillus KEX antibodies are induced and present in antiserum in animals infected with Aspergillus
  • mice exposed to Aspergillus fumigatus were a gift from Dr. Chad
  • mice Jackson mice
  • mice were lightly anesthetized with isoflurane and were administered intratracheally (i.t.) 1 x lO 7 live Aspergillus fumigatus conidia in a volume of 50 ⁇ L of PBS. Starting at day 7, mice were challenged i.t. with 1 ⁇ 10 6 live A. fumigatus conidia in 50 of PBS daily for 5 days, rested for 2 days, and challenged daily for another 3 days. At 24 hours after the final challenge, serum was collected from blood. Uninfected mice were purchased from Taconic Farms Incorporated (Germantown, NY), and serum samples were collected as described above.
  • Aspergillus KEX (AF_KexDS88) was expressed in E. coli and purified. TEV cleaved proteins were resolved and transferred to nitrocellulose membranes as described above. Membranes were then blocked for 2 hours at room temperature with 2% BSA in PBS-T and then were incubated with sera from
  • FIG. 6B is binding of the Aspergillus -infected mouse serum antibodies to the Cryptococcus KEX, Aspergillus KEX, Candida KEX, and Pneumocystis KEXl peptides on the blot.
  • Example 8 Severe asthmatics have lower KEXl titers compared to other subjects with severe lung disease.
  • Standard KEXl ELISA were performed as described in Example 6 on sera collected from a cohort of pediatric patients with severe asthma and from a cohort of pediatric patients with cystic fibrosis (CF). Patients with severe asthma were more likely to have low or undetectable KEXl titers than CF patients.
  • mean anti-KEXl IgG serum titers observed in severe asthmatics were significantly lower than titers observed in CF patients.
  • the present data suggest that low or undetectable titers to Pneumocystis KEXl may be associated with severe asthma and that KEXl antibodies play a role in protection against fungal colonization that can damage the lungs.

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Abstract

L'invention concerne de manière générale des méthodes de traitement ou de prévention d'une infection et/ou d'une maladie associées à différents pathogènes fongiques chez un sujet en ayant besoin, au moyen d'un antisérum isolé généré contre un peptide immunogène d'un pathogène fongique qui contient des anticorps qui protègent de manière croisée le sujet contre une infection et/ou une maladie associées à un ou plusieurs pathogènes fongiques différents. L'antisérum peut être généré contre un peptide Kexine dérivé d'un pathogène fongique parmi Pneumocystis, Aspergillus, Candida ou Cryptococcus. L'antisérum isolé de protection croisée ainsi obtenu peut être utilisé en tant qu'agent thérapeutique pour le traitement ou la protection d'un sujet qui reçoit l'antisérum contre une infection et/ou une maladie associées à de multiples pathogènes fongiques, en plus du pathogène contre lequel l'antisérum est généré. L'invention concerne également des compositions et des kits permettant de détecter ou de quantifier la présence d'anticorps dirigés contre un peptide Kex d'un, deux, trois pathogènes, ou plus, parmi Pneumocystis, Aspergillus, Candida ou Cryptococcus chez un sujet.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11547747B2 (en) 2017-09-27 2023-01-10 University Of Georgia Research Foundation, Inc. Treatment and detection of infection and disease associated with different fungal pathogens
US12453761B2 (en) 2019-01-24 2025-10-28 University Of Georgia Research Foundation, Inc. Treatment and detection of infection and disease associated with different fungal pathogens
WO2021097021A3 (fr) * 2019-11-14 2021-07-15 University Of Georgia Research Foundation, Inc. Traitement et protection contre une infection à aspergillus et une maladie liée à l'aspergillose
US20220401533A1 (en) * 2019-11-14 2022-12-22 University Of Georgia Research Foundation, Inc. Treatment and protection against aspergillus infection and aspergillosis disease

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